title: [Molecular mechanisms of resistance to phosphatidyl inositol 3-kinase inhibitors in triple-negative breast cancer cells].
abstract: OBJECTIVE
  To explore the molecular mechanisms of resistance to phosphatidyl inositol 3-kinase (PI3K) inhibitors in triple-negative breast cancer (TNBC) cells.
  
  
  METHODS
  HCC70 cells (TNBC) were transfected with siFZD7, siWANT5B or siGSK3 using lipofectamine 2000 transfection reagent. The expression levels of key proteins of WNT/β-catenin and PI3K/AKT/mTOR pathways were determined by Western blot analysis. After HCC70, MCF-7 (ER-positive) and SK-BR3 (HER2-positive) cells were treated with PI3K/AKT/mTOR inhibitors, the inhibition rates of cell proliferation were measured by MTT assay, and half maximal inhibitory concentrations (IC50) were calculated. The altered activities of WNT/β-catenin and PI3K/AKT/mTOR proteins were detected by Western blot and luciferase report gene assay, respectively. The nuclear translocation of β-catenin protein was examined by immunofluorescence assay. Xenograft nude mouse model was used to evaluate the tumorigenicity of breast cancer cells treated with BKM120 in vivo. The expression levels of p-LRP6, p-4EBP1 and β-catenin proteins in the tumor tissues were determined by immunohistochemical staining.
  
  
  RESULTS
  The expression levels of FZD7, WANT5B and GSK3 proteins were significantly reduced in the HCC70 cells transfected with the target siRNAs. Meanwhile, the activity of WNT/β-catenin was enhanced and PI3K/AKT/mTOR pathway was inhibited. PI3K/AKT/mTOR inhibitors suppressed MCF-7 and SK-BR3 cell proliferation. The IC50 of GDC-094, BKM120, XL147, perifosine, everolimus, and BEZ235 in MCF-7 cells were 0.46 mmol/L, 1.44 mmol/L, 4.34 mmol/L, 11.35 μmol/L, 53.71 μmol/L and 12.87 μmol/L respectively, and 0.63 mmol/L, 0.58 mmol/L, 3.74 mmol/L, 13.22 μmol/L, 60.00 μmol/L and 11.38 μmol/L in the SK-BR3 cells, respectively. The results of luciferase report gene assay showed that the luciferase activities in HCC70, MCF-7 and SK-BR3 cells treated with BKM120 were 1.75±0.05, 1.13±0.02 and 0.43±0.01, respectively. The luciferase activities in HCC70 and SK-BR3 cells were significantly different from that of the control cells (1.00±0.02, P<0.05). The immunohistochemical analysis showed that BKM120 inhibited mTOR activity, and the enhanced WNT/β-catenin activity reversed the phenotype of inhibitory mTOR induced by BKM120. BKM120 suppressed the tumorigenic ability of MCF-7 and SK-BR3 cells in vivo, but had no effect on cultured HCC70 cells. The immunohistochemical analysis showed nuclear translocation of β-catenin protein and increased expression level of p-LRP-6 protein in transplanted tumor tissues from HCC70 cells treated with BKM120, increased the level of p-LRP-6 protein, and no changes of p-4EBP1 protein expression. However, no nuclear translocation of β-catenin protein and no decrease of p-LRP6 and p-4EBP1 protein levels in the transplanted tumor tissue of MCF-7 cells after treatment with BKM120.
  
  
  CONCLUSIONS
  The triple-negative breast cancer HCC70 cells have drugs-resistance to PI3K inhibitors. The WNT/β-catenin signaling pathway may regulate the PI3K/AKT/mTOR pathway, therefore, inducing the drug-resistance of TNBC cells to PI3K inhibitors.
authors.affiliation: Department of General Surgery, the Rockets Army General hospital of PLA, Beijing 100088, China.
authors.forename: W L
authors.initials: WL
authors.lastname: Zhang
authors.affiliation: Health Team, Public Security Frontier Detachment of Dandong, Dandong 118000, China.
authors.forename: W J
authors.initials: WJ
authors.lastname: Ma
authors.affiliation: Department of General Surgery, the Rockets Army General hospital of PLA, Beijing 100088, China.
authors.forename: S
authors.initials: S
authors.lastname: Chen
authors.affiliation: Department of Pathology, the Rockets Army General hospital of PLA, Beijing 100088, China.
authors.forename: X Z
authors.initials: XZ
authors.lastname: Wu
authors.affiliation: Department of General Surgery, the Rockets Army General hospital of PLA, Beijing 100088, China.
authors.forename: H R
authors.initials: HR
authors.lastname: Zhang
authors.affiliation: Department of General Surgery, the Rockets Army General hospital of PLA, Beijing 100088, China.
authors.forename: J H
authors.initials: JH
authors.lastname: Zhang
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000631:Aminopyridines; D000818:Animals; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019008:Drug Resistance, Neoplasm; D006801:Humans; D007093:Imidazoles; D051379:Mice; D009025:Morpholines; D058539:Phosphatidylinositol 3-Kinase; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D051057:Proto-Oncogene Proteins c-akt; D011804:Quinolines; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases; D064726:Triple Negative Breast Neoplasms; D051176:beta Catenin
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000631:Aminopyridines; D018797:Cell Cycle Proteins; D007093:Imidazoles; D009025:Morpholines; D058539:Phosphatidylinositol 3-Kinase; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D051057:Proto-Oncogene Proteins c-akt; D011804:Quinolines; D058570:TOR Serine-Threonine Kinases; D051176:beta Catenin; C087000:EIF4EBP1 protein, human; C571178:NVP-BKM120; C546842:MTOR protein, human; C491754:AKT3 protein, human; C531198:dactolisib