title: Overexpression of CD44 accompanies acquired tamoxifen resistance in MCF7 cells and augments their sensitivity to the stromal factors, heregulin and hyaluronan.
abstract: BACKGROUND
  Acquired resistance to endocrine therapy in breast cancer is a significant problem with relapse being associated with local and/or regional recurrence and frequent distant metastases. Breast cancer cell models reveal that endocrine resistance is accompanied by a gain in aggressive behaviour driven in part through altered growth factor receptor signalling, particularly involving erbB family receptors. Recently we identified that CD44, a transmembrane cell adhesion receptor known to interact with growth factor receptors, is upregulated in tamoxifen-resistant (TamR) MCF7 breast cancer cells. The purpose of this study was to explore the consequences of CD44 upregulation in an MCF7 cell model of acquired tamoxifen resistance, specifically with respect to the hypothesis that CD44 may influence erbB activity to promote an adverse phenotype.
  
  
  METHODS
  CD44 expression in MCF7 and TamR cells was assessed by RT-PCR, Western blotting and immunocytochemistry. Immunofluorescence and immunoprecipitation studies revealed CD44-erbB associations. TamR cells (± siRNA-mediated CD44 suppression) or MCF7 cells (± transfection with the CD44 gene) were treated with the CD44 ligand, hyaluronon (HA), or heregulin and their in vitro growth (MTT), migration (Boyden chamber and wound healing) and invasion (Matrigel transwell migration) determined. erbB signalling was assessed using Western blotting. The effect of HA on erbB family dimerisation in TamR cells was determined by immunoprecipitation in the presence or absence of CD44 siRNA.
  
  
  RESULTS
  TamR cells overexpressed CD44 where it was seen to associate with erbB2 at the cell surface. siRNA-mediated suppression of CD44 in TamR cells significantly attenuated their response to heregulin, inhibiting heregulin-induced cell migration and invasion. Furthermore, TamR cells exhibited enhanced sensitivity to HA, with HA treatment resulting in modulation of erbB dimerisation, ligand-independent activation of erbB2 and EGFR and induction of cell migration. Overexpression of CD44 in MCF7 cells, which lack endogenous CD44, generated an HA-sensitive phenotype, with HA-stimulation promoting erbB/EGFR activation and migration.
  
  
  CONCLUSIONS
  These data suggest an important role for CD44 in the context of tamoxifen-resistance where it may augment cellular response to erbB ligands and HA, factors that are reported to be present within the tumour microenvironment in vivo. Thus CD44 may present an important determinant of breast cancer progression in the setting of endocrine resistance.
authors.affiliation: Welsh School of Pharmacy, Cardiff University, Wales, UK. HiscoxSE1@cf.ac.uk
authors.forename: Stephen
authors.initials: S
authors.lastname: Hiscox
authors.forename: Bedanta
authors.initials: B
authors.lastname: Baruha
authors.forename: Chris
authors.initials: C
authors.lastname: Smith
authors.forename: Rebecca
authors.initials: R
authors.lastname: Bellerby
authors.forename: Lindy
authors.initials: L
authors.lastname: Goddard
authors.forename: Nicola
authors.initials: N
authors.lastname: Jordan
authors.forename: Zaruhi
authors.initials: Z
authors.lastname: Poghosyan
authors.forename: Robert I
authors.initials: RI
authors.lastname: Nicholson
authors.forename: Peter
authors.initials: P
authors.lastname: Barrett-Lee
authors.forename: Julia
authors.initials: J
authors.lastname: Gee
mesh_terms: D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D066246:ErbB Receptors / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics* / Q000378:metabolism; D006820:Hyaluronic Acid / Q000494:pharmacology*; D061986:MCF-7 Cells; D008856:Microscopy, Fluorescence; D020890:Neuregulin-1 / Q000494:pharmacology*; D055503:Protein Multimerization / Q000187:drug effects; D034622:RNA Interference; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013629:Tamoxifen / Q000494:pharmacology*
chemical_list: D018931:Antineoplastic Agents, Hormonal; D066246:ErbB Receptors; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid; D020890:Neuregulin-1; D018719:Erb-b2 Receptor Tyrosine Kinases; D013629:Tamoxifen