title: erbB3 recruitment of insulin receptor substrate 1 modulates insulin-like growth factor receptor signalling in oestrogen receptor-positive breast cancer cell lines.
abstract: BACKGROUND
  Recently we reported that insulin receptor substrate 1 (IRS-1), classically an adaptor protein for the insulin-like growth factor type I receptor (IGF-IR), associates with the epidermal growth factor receptor in oestrogen receptor (ER)-positive (ER+) tamoxifen-resistant breast cancer cells. In this study, we examined whether IRS-1 also associates with another erbB receptor family member, erbB3, and what impact this might have on IGF-IR signalling in three ER+ breast cancer cell lines.
  
  
  METHODS
  Immunoprecipitation and Western blot analysis were utilised to examine the potential association between erbB3 and IRS-1 in MCF-7, T47D and BT-474 cells in the absence and presence of the erbB3/4 ligand heregulin β1 (HRGβ1). Subsequently, the impact of a selective IGF-IR/IR inhibitor 4-anilino-5-bromo-2-[4-(2-hydroxy-3-(N, N-dimethylamino)propoxy)anilino]pyrimidine on this association and HRGβ1 signalling was assessed in these cell lines. Immunohistochemical analysis of a small cohort of ER+ breast cancer patient samples was also performed to determine the potential clinical relevance of this novel interaction.
  
  
  RESULTS
  Immunoprecipitation and Western blot analysis revealed an interaction between erbB3 and IRS-1 in MCF-7, T47D and BT-474 cells, with HRGβ1 significantly enhancing this recruitment and promoting IRS-1 phosphorylation at Y612. IRS-1 participates in erbB3 signalling in MCF-7 and T47D cells as IRS-1 knockdown impaired HRGβ1 signalling. Importantly, recruitment of IRS-1 by erbB3 reduced IRS-1 association with IGF-IR in MCF-7 and T47D cells, whilst blockade of IGF-IR-enhanced erbB3-IRS-1 interaction and sensitised both cell lines to HRGβ1, allowing HRGβ1 to override IGF-IR blockade. Consequently, suppression of IRS-1 signalling enhanced the effects of IGF-IR inhibition in these cells. This novel interaction may have clinical relevance, as immunohistochemical analysis of a small ER+ breast tumour series revealed significant positive correlations between phosphorylated IRS-1 Y612 expression and total erbB3, phosphorylated Akt and Ki-67 expression.
  
  
  CONCLUSIONS
  IRS-1 can be recruited to IGF-IR and erbB3 in ER+ breast cancer cells, and this provides an adaptive resistance mechanism when these receptors are targeted individually. Consequently, cotargeting IGF-IR and either erbB3 or IRS-1 should prove to be a more effective strategy for the treatment of ER+ breast cancer.
authors.affiliation: Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff, CF10 3NB, UK.
authors.forename: Janice M
authors.initials: JM
authors.lastname: Knowlden
authors.forename: Julia M W
authors.initials: JM
authors.lastname: Gee
authors.forename: Denise
authors.initials: D
authors.lastname: Barrow
authors.forename: John F
authors.initials: JF
authors.lastname: Robertson
authors.forename: Ian O
authors.initials: IO
authors.lastname: Ellis
authors.forename: Robert I
authors.initials: RI
authors.lastname: Nicholson
authors.forename: Iain R
authors.initials: IR
authors.lastname: Hutcheson
mesh_terms: D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004164:Diphosphonates / Q000494:pharmacology; D005260:Female; D006801:Humans; D055504:Insulin Receptor Substrate Proteins / Q000235:genetics / Q000378:metabolism*; D020890:Neuregulin-1 / Q000494:pharmacology; D010766:Phosphorylation; D034741:RNA, Small Interfering; D020893:Receptor, ErbB-3 / Q000235:genetics / Q000378:metabolism*; D017526:Receptor, IGF Type 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: C514497:4-amino-1-hydroxybutylidene-1,1-diphosphonate; D004164:Diphosphonates; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D020890:Neuregulin-1; D034741:RNA, Small Interfering; D011960:Receptors, Estrogen; C087230:heregulin beta1; D020893:Receptor, ErbB-3; D017526:Receptor, IGF Type 1