title: Nicastrin and Notch4 drive endocrine therapy resistance and epithelial to mesenchymal transition in MCF7 breast cancer cells.
abstract: BACKGROUND
  Resistance to anti-estrogen therapies is a major cause of disease relapse and mortality in estrogen receptor alpha (ERα)-positive breast cancers. Tamoxifen or estrogen withdrawal increases the dependence of breast cancer cells on Notch signalling. Here, we investigated the contribution of Nicastrin and Notch signalling in endocrine-resistant breast cancer cells.
  
  
  METHODS
  We used two models of endocrine therapies resistant (ETR) breast cancer: tamoxifen-resistant (TamR) and long-term estrogen-deprived (LTED) MCF7 cells. We evaluated the migratory and invasive capacity of these cells by Transwell assays. Expression of epithelial to mesenchymal transition (EMT) regulators as well as Notch receptors and targets were evaluated by real-time PCR and western blot analysis. Moreover, we tested in vitro anti-Nicastrin monoclonal antibodies (mAbs) and gamma secretase inhibitors (GSIs) as potential EMT reversal therapeutic agents. Finally, we generated stable Nicastrin overexpessing MCF7 cells and evaluated their EMT features and response to tamoxifen.
  
  
  RESULTS
  We found that ETR cells acquired an epithelial to mesenchymal transition (EMT) phenotype and displayed increased levels of Nicastrin and Notch targets. Interestingly, we detected higher level of Notch4 but lower levels of Notch1 and Notch2 suggesting a switch to signalling through different Notch receptors after acquisition of resistance. Anti-Nicastrin monoclonal antibodies and the GSI PF03084014 were effective in blocking the Nicastrin/Notch4 axis and partially inhibiting the EMT process. As a result of this, cell migration and invasion were attenuated and the stem cell-like population was significantly reduced. Genetic silencing of Nicastrin and Notch4 led to equivalent effects. Finally, stable overexpression of Nicastrin was sufficient to make MCF7 unresponsive to tamoxifen by Notch4 activation.
  
  
  CONCLUSIONS
  ETR cells express high levels of Nicastrin and Notch4, whose activation ultimately drives invasive behaviour. Anti-Nicastrin mAbs and GSI PF03084014 attenuate expression of EMT molecules reducing cellular invasiveness. Nicastrin overexpression per se induces tamoxifen resistance linked to acquisition of EMT phenotype. Our finding suggest that targeting Nicastrin and/or Notch4 warrants further clinical evaluation as valid therapeutic strategies in endocrine-resistant breast cancer.
authors.forename: Ylenia
authors.initials: Y
authors.lastname: Lombardo
authors.forename: Monica
authors.initials: M
authors.lastname: Faronato
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Filipovic
authors.forename: Valentina
authors.initials: V
authors.lastname: Vircillo
authors.forename: Luca
authors.initials: L
authors.lastname: Magnani
authors.forename: R Charles
authors.initials: RC
authors.lastname: Coombes
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000473:pathology; D051927:CD24 Antigen / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D058750:Epithelial-Mesenchymal Transition / Q000502:physiology*; D047628:Estrogen Receptor alpha / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D061986:MCF-7 Cells; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D009361:Neoplasm Invasiveness; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering; D051881:Receptor, Notch1 / Q000378:metabolism; D051883:Receptor, Notch2 / Q000378:metabolism; D000074562:Receptor, Notch4; D051880:Receptors, Notch / Q000235:genetics* / Q000378:metabolism; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology; D018874:Spheroids, Cellular; D013629:Tamoxifen / Q000494:pharmacology*; D013764:Tetrahydronaphthalenes / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014633:Valine / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D018931:Antineoplastic Agents, Hormonal; D051927:CD24 Antigen; C497889:CD24 protein, human; C497483:CD44 protein, human; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D018960:Hyaluronan Receptors; D008562:Membrane Glycoproteins; C497761:NOTCH1 protein, human; C497764:NOTCH2 protein, human; C106075:NOTCH4 protein, human; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D051881:Receptor, Notch1; D051883:Receptor, Notch2; D000074562:Receptor, Notch4; D051880:Receptors, Notch; D020845:Selective Estrogen Receptor Modulators; D013764:Tetrahydronaphthalenes; C413604:nicastrin protein; D013629:Tamoxifen; D053829:Amyloid Precursor Protein Secretases; D014633:Valine; C550722:nirogacestat