Epidermal growth factor receptor, c-erbB2 and c-erbB3 receptor interaction, and related cell cycle kinetics of SK-BR-3 and BT474 breast carcinoma cells

Cytometry. 2001 Aug 1;44(4):338-48. doi: 10.1002/1097-0320(20010801)44:4<338::aid-cyto1125>3.0.co;2-v.

Abstract

Background: Receptors belonging to the epidermal growth factor receptor (EGFR) family transfer extracellular signals by homotypic and heterotypic receptor interaction and cross-activation. Cell differentiation, death, and proliferation are regulated via these receptor-tyrosine-kinases. However, the initial mechanisms that lead to signal specificity and diversity, which cause a defined cellular response, are incompletely understood. We investigated the recruitment of receptor complexes in two c-erbB2-overexpressing breast carcinoma cell lines, SK-BR-3 and BT474, after ligand binding and its effects on intracellular signal transduction and cell cycle regulation.

Methods: In order to analyze the coaggregation of receptors on the cell surface induced by specific growth factor treatment, we used the flow cytometric Foerster-type fluorescence resonance energy transfer (FRET) technique. Cell cycle kinetics were monitored flow cytometrically via the anti-BrdU technique and acitivation of intracellular signal cascades was analyzed by Western blotting.

Results: After stimulation with EGF BT474, but not SK-BR-3, cells formed EGFR/c-erbB2 receptor complexes. Neither EGF nor heregulin (HRG) induced c-erbB2/c-erbB3 receptor complexes in BT474. However, SK-BR-3 cells exhibited a high amount of c-erbB2/c-erbB3 heterodimers even without growth factor stimulation which could be elevated after prolonged EGF and HRG treatment. In both cell lines, mitogen-activated protein kinase (MAPK) phosphorylation was detectable after short-term and prolonged EGF and HRG treatment. However, only SK-BR-3 cells showed a constitutive activation of both protein kinase B (PKB)/Akt and MAPK signaling pathways. Growth factor treatment caused an amplified PKB/Akt activation in this cell line. The induction of EGFR/c-erbB2 complexes in BT474 was associated with shortening of the G1-phase of the cell cycle. In contrast, the concurrent activation of MAPK and PKB/Akt by EGF treatment led to an inhibition of proliferation in SK-BR-3 and can be attributed to missing EGFR/c-erbB2 heterodimers. HRG was a strong stimulator of proliferation in both cell lines.

Conclusions: We show that in the presence of identical amounts of c-erbB2 receptors, the ligand-induced cellular response differs significantly. These differences were mediated by variances in signal transduction, most likely due to different recruitment of heterotypic receptor complexes. Overall, there is strong evidence that c-erbB2 receptor overexpression in breast cancer cells is an insufficient marker to determine cellular response in terms of cell proliferation. 2001.

MeSH terms

  • Breast Neoplasms / metabolism*
  • Breast Neoplasms / pathology*
  • Cell Cycle / drug effects
  • Cell Cycle / physiology
  • Cell Division / drug effects
  • Cell Division / physiology
  • Epidermal Growth Factor / metabolism*
  • Epidermal Growth Factor / pharmacology
  • ErbB Receptors / metabolism*
  • Female
  • G1 Phase / drug effects
  • G1 Phase / physiology
  • Humans
  • Macromolecular Substances
  • Mitogen-Activated Protein Kinase Kinases / drug effects
  • Mitogen-Activated Protein Kinase Kinases / metabolism
  • Neuregulin-1 / pharmacology
  • Protein Binding / drug effects
  • Protein Binding / physiology
  • Protein-Serine-Threonine Kinases*
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-akt
  • Receptor, ErbB-2 / metabolism*
  • Receptor, ErbB-3 / metabolism*
  • Signal Transduction
  • Tumor Cells, Cultured

Substances

  • Macromolecular Substances
  • Neuregulin-1
  • Proto-Oncogene Proteins
  • Epidermal Growth Factor
  • ErbB Receptors
  • Receptor, ErbB-2
  • Receptor, ErbB-3
  • AKT1 protein, human
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • Mitogen-Activated Protein Kinase Kinases