title: Profiling the tyrosine phosphoproteome of different mouse mammary tumour models reveals distinct, model-specific signalling networks and conserved oncogenic pathways.
abstract: BACKGROUND
  Although aberrant tyrosine kinase signalling characterises particular breast cancer subtypes, a global analysis of tyrosine phosphorylation in mouse models of breast cancer has not been undertaken to date. This may identify conserved oncogenic pathways and potential therapeutic targets.
  
  
  METHODS
  We applied an immunoaffinity/mass spectrometry workflow to three mouse models: murine stem cell virus-Neu, expressing truncated Neu, the rat orthologue of human epidermal growth factor receptor 2, Her2 (HER2); mouse mammary tumour virus-polyoma virus middle T antigen (PyMT); and the p53-/- transplant model (p53). Pathways and protein-protein interaction networks were identified by bioinformatics analysis. Molecular mechanisms underpinning differences in tyrosine phosphorylation were characterised by Western blot analysis and array comparative genomic hybridisation. The functional role of mesenchymal-epithelial transition factor (Met) in a subset of p53-null tumours was interrogated using a selective tyrosine kinase inhibitor (TKI), small interfering RNA (siRNA)-mediated knockdown and cell proliferation assays.
  
  
  RESULTS
  The three models could be distinguished on the basis of tyrosine phosphorylation signatures and signalling networks. HER2 tumours exhibited a protein-protein interaction network centred on avian erythroblastic leukaemia viral oncogene homologue 2 (Erbb2), epidermal growth factor receptor and platelet-derived growth factor receptor α, and they displayed enhanced tyrosine phosphorylation of ERBB receptor feedback inhibitor 1. In contrast, the PyMT network displayed significant enrichment for components of the phosphatidylinositol 3-kinase signalling pathway, whereas p53 tumours exhibited increased tyrosine phosphorylation of Met and components or regulators of the cytoskeleton and shared signalling network characteristics with basal and claudin-low breast cancer cells. A subset of p53 tumours displayed markedly elevated cellular tyrosine phosphorylation and Met expression, as well as Met gene amplification. Treatment of cultured p53-null cells exhibiting Met amplification with a selective Met TKI abrogated aberrant tyrosine phosphorylation and blocked cell proliferation. The effects on proliferation were recapitulated when Met was knocked down using siRNA. Additional subtypes of p53 tumours exhibited increased tyrosine phosphorylation of other oncogenes, including Peak1/SgK269 and Prex2.
  
  
  CONCLUSIONS
  This study provides network-level insights into signalling in the breast cancer models utilised and demonstrates that comparative phosphoproteomics can identify conserved oncogenic signalling pathways. The Met-amplified, p53-null tumours provide a new preclinical model for a subset of triple-negative breast cancers.
authors.forename: Naveid A
authors.initials: NA
authors.lastname: Ali
authors.forename: Jianmin
authors.initials: J
authors.lastname: Wu
authors.forename: Falko
authors.initials: F
authors.lastname: Hochgräfe
authors.forename: Howard
authors.initials: H
authors.lastname: Chan
authors.forename: Radhika
authors.initials: R
authors.lastname: Nair
authors.forename: Sunny
authors.initials: S
authors.lastname: Ye
authors.forename: Luxi
authors.initials: L
authors.lastname: Zhang
authors.forename: Ruth J
authors.initials: RJ
authors.lastname: Lyons
authors.forename: Mark
authors.initials: M
authors.lastname: Pinese
authors.forename: Hong Ching
authors.initials: HC
authors.lastname: Lee
authors.forename: Nicola
authors.initials: N
authors.lastname: Armstrong
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Ormandy
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Clark
authors.forename: Alexander
authors.initials: A
authors.lastname: Swarbrick
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Daly
mesh_terms: D000818:Animals; D005260:Female; D018628:Gene Dosage; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009368:Neoplasm Transplantation; D009857:Oncogenes; D010750:Phosphoproteins / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism*; D060066:Protein Interaction Maps; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D020543:Proteome / Q000378:metabolism*; D019859:Proto-Oncogene Proteins c-met / Q000037:antagonists & inhibitors; D013450:Sulfones / Q000494:pharmacology
chemical_list: C480541:5-((2,6-dichlorobenzyl)sulfonyl)-3-((3,5-dimethyl-4-((2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl)carbonyl)-1H-pyrrol-2-yl)methylene)-1,3-dihydro-2H-indol-2-one; D007211:Indoles; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D020543:Proteome; D013450:Sulfones; D019000:Phosphotyrosine; D019859:Proto-Oncogene Proteins c-met