title: Targeting treatment-resistant breast cancer stem cells with FKBPL and its peptide derivative, AD-01, via the CD44 pathway.
abstract: OBJECTIVE
  FK506-binding protein like (FKBPL) and its peptide derivative, AD-01, have already shown tumor growth inhibition and CD44-dependent antiangiogenic activity. Here, we explore the ability of AD-01 to target CD44-positive breast cancer stem cells (BCSC).
  
  
  METHODS
  Mammosphere assays and flow cytometry were used to analyze the effect of FKBPL overexpression/knockdown and AD-01 treatment ± other anticancer agents on BCSCs using breast cancer cell lines (MCF-7/MDA-231/ZR-75), primary patient samples, and xenografts. Delays in tumor initiation were evaluated in vivo. The anti-stem cell mechanisms were determined using clonogenic assays, quantitative PCR (qPCR), and immunofluorescence.
  
  
  RESULTS
  AD-01 treatment was highly effective at inhibiting the BCSC population by reducing mammosphere-forming efficiency and ESA(+)/CD44(+)/CD24(-) or aldehyde dehydrogenase (ALDH)(+) cell subpopulations in vitro and tumor initiation in vivo. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed; mammospheres were completely eradicated by the third generation. The mechanism seems to be due to AD-01-mediated BCSC differentiation shown by a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones; the stem cell markers, Nanog, Oct4, and Sox2, were also significantly reduced. Furthermore, we showed additive inhibitory effects when AD-01 was combined with the Notch inhibitor, DAPT. AD-01 was also able to abrogate a chemo- and radiotherapy-induced enrichment in BCSCs. Finally, FKBPL knockdown led to an increase in Nanog/Oct4/Sox2 and an increase in BCSCs, highlighting a role for endogenous FKBPL in stem cell signaling.
  
  
  CONCLUSIONS
  AD-01 has dual antiangiogenic and anti-BCSC activity, which will be advantageous as this agent enters clinical trial.
authors.affiliation: School of Pharmacy, Queen's University Belfast, Belfast, United Kingdom.
authors.forename: Lana
authors.initials: L
authors.lastname: McClements
authors.forename: Anita
authors.initials: A
authors.lastname: Yakkundi
authors.forename: Angelos
authors.initials: A
authors.lastname: Papaspyropoulos
authors.forename: Hannah
authors.initials: H
authors.lastname: Harrison
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Ablett
authors.forename: Puthen V
authors.initials: PV
authors.lastname: Jithesh
authors.forename: Hayley D
authors.initials: HD
authors.lastname: McKeen
authors.forename: Rachel
authors.initials: R
authors.lastname: Bennett
authors.forename: Christopher
authors.initials: C
authors.lastname: Donley
authors.forename: Adrien
authors.initials: A
authors.lastname: Kissenpfennig
authors.forename: Stuart
authors.initials: S
authors.lastname: McIntosh
authors.forename: Helen O
authors.initials: HO
authors.lastname: McCarthy
authors.forename: Eric
authors.initials: E
authors.lastname: O'Neill
authors.forename: Robert B
authors.initials: RB
authors.lastname: Clarke
authors.forename: Tracy
authors.initials: T
authors.lastname: Robson
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000401:mortality; D019008:Drug Resistance, Neoplasm; D005260:Female; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism*; D020104:Immunophilins / Q000378:metabolism / Q000494:pharmacology*; D061986:MCF-7 Cells; D051379:Mice; D016513:Mice, SCID; D014411:Neoplastic Stem Cells / Q000187:drug effects*; D011836:Radiation Tolerance; D051880:Receptors, Notch / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction; D018874:Spheroids, Cellular / Q000187:drug effects; D022021:Tacrolimus Binding Proteins; D047368:Tumor Burden / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays
chemical_list: C586123:AD-01 peptide; D000970:Antineoplastic Agents; C497483:CD44 protein, human; C400101:FKBPL protein, human; D018960:Hyaluronan Receptors; D051880:Receptors, Notch; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins