title: High basal Wnt signaling is further induced by PI3K/mTor inhibition but sensitive to cSRC inhibition in mammary carcinoma cell lines with HER2/3 overexpression.
abstract: BACKGROUND
  Elevated basal, ligand-independent, Wnt signaling in some canine breast cancer cells is not caused by classical mutations in APC, β-Catenin or GSK3β but, at least partially, by enhanced LEF1 expression. We examined the expression and function of EGFR/HER-regulated pathways on the ligand-independent Wnt signaling.
  
  
  METHODS
  Twelve canine mammary tumor cell lines with previously reported differential basal Wnt activity were used. The expression levels of genes related to EGF-signaling were analyzed by cluster analysis. Cell lines with a combined overexpression of EGF-related genes and enhanced basal Wnt activity were treated with PI3K/mTor or cSRC inhibitors or transfected with a construct expressing wild-type PTEN. Subsequently, effects were measured on Wnt activity, cell proliferation, gene expression and protein level.
  
  
  RESULTS
  High basal Wnt/LEF1 activity was associated with overexpression of HER2/3, ID1, ID2, RAC1 and HSP90 together with low to absent cMET and PTEN mRNA expression, suggesting a connection between Wnt- and HER-signaling pathways. Inhibition of the HER-regulated PI3K/mTor pathway using the dual PI3K/mTor inhibitor BEZ235 or the mTor inhibitor Everolimus® resulted in reduced cell proliferation. In the cell line with high basal Wnt activity, however, an unexpected further increased Wnt activity was found that could be greatly reduced after inhibition of the HER-regulated cSRC activity. Inhibition of the PI3K/mTor pathway was associated with enhanced expression of β-Catenin, Axin2, MUC1, cMET, EGFR and HER2 and a somewhat increased β-Catenin protein content, whereas cSRC inhibition was associated with slightly enhanced HER3 and SLUG mRNA expression. A high protein expression of HER3 was found only in a cell line with high basal Wnt activity.
  
  
  CONCLUSIONS
  High basal Wnt activity in some mammary cancer cell lines is associated with overexpression of HER-receptor related genes and HER3 protein, and the absence of PTEN. Inhibition of the PI3K/mTor pathway further stimulated, however, canonical Wnt signaling, whereas the inhibitory effect with the cSRC inhibitor Src-I1 on the Wnt activity further suggested a connection between Wnt and HER2/3-signaling.
authors.affiliation: Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584, CM, Utrecht, The Netherlands. e.p.m.timmermans@uu.nl.
authors.forename: Elpetra P M
authors.initials: EP
authors.lastname: Timmermans-Sprang
authors.affiliation: Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584, CM, Utrecht, The Netherlands. a.gracanin@uu.nl.
authors.forename: Ana
authors.initials: A
authors.lastname: Gracanin
authors.affiliation: Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584, CM, Utrecht, The Netherlands. j.a.mol@uu.nl.
authors.forename: Jan A
authors.initials: JA
authors.lastname: Mol
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004285:Dogs; D000068338:Everolimus / Q000494:pharmacology*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D007093:Imidazoles / Q000494:pharmacology*; D015674:Mammary Neoplasms, Animal / Q000235:genetics* / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000235:genetics; D011804:Quinolines / Q000494:pharmacology*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D020893:Receptor, ErbB-3 / Q000235:genetics* / Q000378:metabolism; D060449:Wnt Signaling Pathway / Q000187:drug effects
chemical_list: D000068338:Everolimus; D007093:Imidazoles; D016392:Proto-Oncogene Proteins pp60(c-src); D011804:Quinolines; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; C531198:dactolisib