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.
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.
Brockhoff
G
G
Institute of Pathology, University Regensburg, Regensberg, Germany. Gero.Brockhoff@klinik.uni-regensburg.de
Heiss
P
P
Schlegel
J
J
Hofstaedter
F
F
Knuechel
R
R
D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D004815:Epidermal Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D005260:Female; D016193:G1 Phase / Q000187:drug effects / Q000502:physiology; D006801:Humans; D046911:Macromolecular Substances; D020929:Mitogen-Activated Protein Kinase Kinases / Q000187:drug effects / Q000378:metabolism; D020890:Neuregulin-1 / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D020893:Receptor, ErbB-3 / Q000378:metabolism*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D046911:Macromolecular Substances; D020929:Mitogen-Activated Protein Kinase Kinases; D020890:Neuregulin-1; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; C494918:AKT1 protein, human