title: Chemotherapy induces Notch1-dependent MRP1 up-regulation, inhibition of which sensitizes breast cancer cells to chemotherapy.
abstract: BACKGROUND
  Multi-drug Resistance associated Protein-1 (MRP1) can export chemotherapeutics from cancer cells and is implicated in chemoresistance, particularly as is it known to be up-regulated by chemotherapeutics. Our aims in this study were to determine whether activation of Notch signalling is responsible for chemotherapy-induced MRP1 expression Notch in breast cancers, and whether this pathway can be manipulated with an inhibitor of Notch activity.
  
  
  METHODS
  MRP1 and Notch1 were investigated in 29 patients treated with neoadjuvant chemotherapy (NAC) for breast cancer, using immunohistochemistry on matched biopsy (pre-NAC) and surgical samples (post-NAC). Breast epithelial cell cultures (T47D, HB2) were treated with doxorubicin in the presence and absence of functional Notch1, and qPCR, siRNA, Western blots, ELISAs and flow-cytometry were used to establish interactions.
  
  
  RESULTS
  In clinical samples, Notch1 was activated by neoadjuvant chemotherapy (Wilcoxon signed-rank p < 0.0001) and this correlated with induction of MRP1 expression (rho = 0.6 p = 0.0008). In breast cell lines, doxorubicin induced MRP1 expression and function (non-linear regression p < 0.004). In the breast cancer line T47D, doxorubicin activated Notch1 and, critically, inhibition of Notch1 activation with the γ-secretase inhibitor DAPT abolished the doxorubicin-induced increase in MRP1 expression and function (t-test p < 0.05), resulting in enhanced cellular retention of doxorubicin and increased doxorubicin-induced apoptosis (t-test p = 0.0002). In HB2 cells, an immortal but non-cancer derived breast cell line, Notch1-independent MRP1 induction was noted and DAPT did not enhance doxorubicin-induced apoptosis.
  
  
  CONCLUSIONS
  Notch inhibitors may have potential in sensitizing breast cancer cells to chemotherapeutics and therefore in tackling chemoresistance.
authors.affiliation: School of Medicine, University of Leeds, Leeds, UK. B.Kim@leeds.ac.uk.|Department of Breast Surgery, Leeds Teaching Hospitals NHS Trust, Leeds, UK. B.Kim@leeds.ac.uk.
authors.forename: Baek
authors.initials: B
authors.lastname: Kim
authors.affiliation: School of Medicine, University of Leeds, Leeds, UK. S.L.Stephen@leeds.ac.uk.
authors.forename: Sam L
authors.initials: SL
authors.lastname: Stephen
authors.affiliation: School of Medicine, University of Leeds, Leeds, UK. A.M.Hanby@leeds.ac.uk.|Department of Histopathology, Leeds Teaching Hospitals NHS Trust, Leeds, UK. A.M.Hanby@leeds.ac.uk.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Hanby
authors.affiliation: Department of Breast Surgery, Leeds Teaching Hospitals NHS Trust, Leeds, UK. kieran.horgan@nhs.net.
authors.forename: Kieran
authors.initials: K
authors.lastname: Horgan
authors.affiliation: School of Medicine, University of Leeds, Leeds, UK. S.L.Perry@leeds.ac.uk.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Perry
authors.affiliation: School of Medicine, University of Leeds, Leeds, UK. jewels_555@msn.com.
authors.forename: Julie
authors.initials: J
authors.lastname: Richardson
authors.affiliation: School of Medicine, University of Leeds, Leeds, UK. E.A.Roundhill@leeds.ac.uk.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Roundhill
authors.affiliation: School of Medicine, University of Leeds, Leeds, UK. E.M.A.Valleley@leeds.ac.uk.
authors.forename: Elizabeth M A
authors.initials: EM
authors.lastname: Valleley
authors.affiliation: School of Medicine, University of Leeds, Leeds, UK. E.T.Verghese@leeds.ac.uk.|Department of Histopathology, Leeds Teaching Hospitals NHS Trust, Leeds, UK. E.T.Verghese@leeds.ac.uk.
authors.forename: Eldo T
authors.initials: ET
authors.lastname: Verghese
authors.affiliation: Department of Histopathology, Leeds Teaching Hospitals NHS Trust, Leeds, UK. bethwilliams35@hotmail.com.
authors.forename: Bethany J
authors.initials: BJ
authors.lastname: Williams
authors.affiliation: School of Food Science and Nutrition, University of Leeds, Leeds, UK. J.L.Thorne@leeds.ac.uk.
authors.forename: James L
authors.initials: JL
authors.lastname: Thorne
authors.affiliation: School of Medicine, University of Leeds, Leeds, UK. t.hughes@leeds.ac.uk.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Hughes
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000627:therapeutic use*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D008875:Middle Aged; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D020360:Neoadjuvant Therapy; D060888:Real-Time Polymerase Chain Reaction; D051881:Receptor, Notch1 / Q000378:metabolism*; D015398:Signal Transduction; D015854:Up-Regulation / Q000187:drug effects*; D055815:Young Adult
chemical_list: D000970:Antineoplastic Agents; D004317:Doxorubicin; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D051881:Receptor, Notch1; C497761:NOTCH1 protein, human; C421109:multidrug resistance-associated protein 1