title: Cytoplasmic levels of cFLIP determine a broad susceptibility of breast cancer stem/progenitor-like cells to TRAIL.
abstract: BACKGROUND
  The clinical application of TRAIL receptor agonists as a novel cancer therapy has been tempered by heterogeneity in tumour responses. This is illustrated in breast cancer, where TRAIL is cytotoxic in cell lines of mesenchymal origin but refractory in lines with an epithelial-like phenotype. However, it is now evident that intra-tumour heterogeneity includes a minority subpopulation of tumour-initiating stem/progenitor-like cells (CSCs) that possess mesenchymal characteristics. We hypothesised therefore that TRAIL may target these phenotypically distinct CSC-like cells that are common to most - if not all - breast cancers, thus impacting on the source of malignancy in a much broader range of breast tumour subtypes than previously envisaged.
  
  
  METHODS
  We used colony formation, tumoursphere, flow cytometry and xenograft tumour initiation assays to observe the TRAIL sensitivity of CSC-like cells in a panel of two mesenchymal-like (TRAIL-sensitive) and four epithelial-like (TRAIL-resistant) breast cancer cell lines. Subcellular levels of the endogenous TRAIL inhibitor, cFLIP, were determined by western blot and immunofluorescence microscopy. The effect of the subcellular redistribution of cFLIP on TRAIL sensitivity and Wnt signalling was determined using cFLIP localisation mutants and the TOPFlash reporter assay respectively.
  
  
  RESULTS
  TRAIL universally suppressed the clonal expansion of stem/progenitors in all six of the breast cancer cell lines tested, irrespective of their phenotype or overall sensitivity to TRAIL. A concomitant reduction in tumour initiation was confirmed in the TRAIL-resistant epithelial cell line, MCF-7, following serial dilution xenotransplantation. Furthermore TRAIL sensitivity of breast CSCs was inversely proportional to the relative cytoplasmic levels of cFLIP while overexpression of cFLIP in the cytosol using subcellular localization mutants of cFLIP protected these cells from cytotoxicity. The accumulation of nuclear cFLIP on the other hand did not influence TRAIL cytotoxicity but instead promoted Wnt-dependent signalling.
  
  
  CONCLUSIONS
  These data propose a novel role for TRAIL as a selective CSC agent with a broad specificity for both epithelial and mesenchymal breast tumour subtypes. Furthermore we identify a dual role for cFLIP in the maintenance of breast CSC viability, dependent upon its subcellular distribution.
authors.affiliation: European Cancer Stem Cell Research Institute Cardiff University School of Biosciences, Haydn Ellis Building, Maindy Road, Cardiff, CF24 4HQ, UK. frenchr@cardiff.ac.uk.
authors.forename: Rhiannon
authors.initials: R
authors.lastname: French
authors.affiliation: European Cancer Stem Cell Research Institute Cardiff University School of Biosciences, Haydn Ellis Building, Maindy Road, Cardiff, CF24 4HQ, UK. haywardo@cardiff.ac.uk.
authors.forename: Olivia
authors.initials: O
authors.lastname: Hayward
authors.affiliation: European Cancer Stem Cell Research Institute Cardiff University School of Biosciences, Haydn Ellis Building, Maindy Road, Cardiff, CF24 4HQ, UK. jonessr15@cf.ac.uk.
authors.forename: Samuel
authors.initials: S
authors.lastname: Jones
authors.affiliation: European Cancer Stem Cell Research Institute Cardiff University School of Biosciences, Haydn Ellis Building, Maindy Road, Cardiff, CF24 4HQ, UK. yangw5@cf.ac.uk.
authors.forename: William
authors.initials: W
authors.lastname: Yang
authors.affiliation: European Cancer Stem Cell Research Institute Cardiff University School of Biosciences, Haydn Ellis Building, Maindy Road, Cardiff, CF24 4HQ, UK. clarksonr@cf.ac.uk.
authors.forename: Richard
authors.initials: R
authors.lastname: Clarkson
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D003593:Cytoplasm / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D005260:Female; D006801:Humans; D008819:Mice, Nude; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000502:physiology; D021381:Protein Transport; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology*; D060449:Wnt Signaling Pathway; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D053221:TNF-Related Apoptosis-Inducing Ligand