title: Profiling the HER3/PI3K pathway in breast tumors using proximity-directed assays identifies correlations between protein complexes and phosphoproteins.
abstract: BACKGROUND
  The identification of patients for targeted antineoplastic therapies requires accurate measurement of therapeutic targets and associated signaling complexes. HER3 signaling through heterodimerization is an important growth-promoting mechanism in several tumor types and may be a principal resistance mechanism by which EGFR and HER2 expressing tumors elude targeted therapies. Current methods that can study these interactions are inadequate for formalin-fixed, paraffin-embedded (FFPE) tumor samples.
  
  
  RESULTS
  Herein, we describe a panel of proximity-directed assays capable of measuring protein-interactions and phosphorylation in FFPE samples in the HER3/PI3K/Akt pathway and examine the capability of these assays to inform on the functional state of the pathway. We used FFPE breast cancer cell line and tumor models for this study. In breast cancer cell lines we observe both ligand-dependent and independent activation of the pathway and strong correlations between measured activation of key analytes. When selected cell lines are treated with HER2 inhibitors, we not only observe the expected molecular effects based on mechanism of action knowledge, but also novel effects of HER2 inhibition on key targets in the HER receptor pathway. Significantly, in a xenograft model of delayed tumor fixation, HER3 phosphorylation is unstable, while alternate measures of pathway activation, such as formation of the HER3PI3K complex is preserved. Measurements in breast tumor samples showed correlations between HER3 phosphorylation and receptor interactions, obviating the need to use phosphorylation as a surrogate for HER3 activation.
  
  
  CONCLUSIONS
  This assay system is capable of quantitatively measuring therapeutically relevant responses and enables molecular profiling of receptor networks in both preclinical and tumor models.
authors.affiliation: Department of Oncology, Monogram Biosciences, South San Francisco, California, United States of America. amukherjee@monogrambio.com
authors.forename: Ali
authors.initials: A
authors.lastname: Mukherjee
authors.forename: Youssouf
authors.initials: Y
authors.lastname: Badal
authors.forename: Xuan-Thao
authors.initials: XT
authors.lastname: Nguyen
authors.forename: Johanna
authors.initials: J
authors.lastname: Miller
authors.forename: Ahmed
authors.initials: A
authors.lastname: Chenna
authors.forename: Hasan
authors.initials: H
authors.lastname: Tahir
authors.forename: Alicia
authors.initials: A
authors.lastname: Newton
authors.forename: Gordon
authors.initials: G
authors.lastname: Parry
authors.forename: Stephen
authors.initials: S
authors.lastname: Williams
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D006801:Humans; D051379:Mice; D046912:Multiprotein Complexes / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D020893:Receptor, ErbB-3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction; D014183:Transplantation, Heterologous
chemical_list: D046912:Multiprotein Complexes; D010750:Phosphoproteins; D019869:Phosphatidylinositol 3-Kinases; D020893:Receptor, ErbB-3