176P - HER-3 targeting affects the dimerization pattern of EGFR family members in breast carcinomas (BC)

Date 28 September 2014
Event ESMO 2014
Session Poster Display session
Topics Anti-Cancer Agents & Biologic Therapy
Breast Cancer
Translational Research
Presenter Michalis Karamouzis
Citation Annals of Oncology (2014) 25 (suppl_4): iv58-iv84. 10.1093/annonc/mdu326
Authors M.V. Karamouzis1, G. Dalagiorgou2, U. Georgopoulou3, M. Kontos4, A.G. Papavassiliou5
  • 1Molecular Oncology Unit, Department Of Biological Chemistry, Medical School, University of Athens, 10676 - Athens/GR
  • 2Molecular Oncology Unit, Department Of Biological Chemistry, Medical School, University of Athens, 11527 - Athens/GR
  • 3Laboratory Of Virology, HELLENIC PASTEUR INSTITUTE, 11527 - Athens/GR
  • 4First Department Of Surgery, Laiko Hospital, University of Athens, 11527 - Athens/GR
  • 5Department Of Biological Chemistry, University of Athens, School of Medicine, 11527 - Athens/GR

Abstract

Aim

Many BC patients present intrinsic or acquired resistance to anti-HER-2 directed therapies, mainly attributed to HER-3. Our goal was to evaluate the dimerization pattern of EGFR, HER2/3/4 with or without the addition of heregulin (Hg) and HER-3 targeting agents.

Methods

MCF7 and SKBR3 cell lines were used. Dimerization patterns were studied using proximity ligation assay and immunofluorescence, where proteins in proximity can be visualized by combining classical immunocytochemistry with rolling circle amplification. Concentrations of used reagents were: HRG1-ß1 10mM (R&D Systems), Pertuzumab (P) 30mM (Genetech), U3 (U) inhibitor 30mM (U3 Pharma). After blocking, cells were incubated with combination of primary antibodies (antiHER2/HER3, antiHER2/HER4, antiEGFR/HER3, anti-EGFR/HER4) in a preheated humidity chamber for 1 h at 37°C. Cells were then incubated with the PLA probes diluted 1:5 in antibody diluent (Olink Bioscience, Uppsala, Sweden) in a humidified chamber for 1 h at 37°C. Subsequent hybridization, ligation, amplification, and detection were performed as per manufacturer's instruction. Fluorescence images were acquired using a Zeiss Axiovert microscope (Carl Zeiss Microscopy, Thornwood, NY). Data analysis was done using Duolink ImageTool Software that has been developed for quantification of PLA signals.

Results

In MCF7 cells (PLA signals scale 0-12), Hg addition increased dimmers EGFR/HER4 (9,7 vs 5,13), EGFR/HER3 (8,07 vs 3,36) and less HER2/HER3 (4,12 vs 3,35). The addition of Hg and P resulted in EGFR/HER4 4,67, EGFR/HER3 2,46, HER2/HER3 3,0 and Hg and U resulted in EGFR/HER4 0,6, EGFR/HER3 0,6, HER2/HER3 0,6. In SKBR3 cells (PLA signals scale 0-60), Hg addition increased dimmers EGFR/HER4 (60 vs 40), HER2/HER3 (26,93 vs 15,92) and less EGFR/HER3 (16 vs 10,6). The addition of Hg and P resulted in EGFR/HER4 17,0, HER2/HER3 19,9, EGFR/HER3 12,5 and Hg and U resulted in EGFR/HER4 0,7, HER2/HER3 0,7, EGFR/HER3 0,7. All images and data analysis will be presented in full detail.

Conclusions

The addition of Hg enhances HER3-based dimmer formation, while HER-3 targeting agents lower HER3-containing dimmer formation. The technique can be used in paraffin-embedded BC specimens and may identify patient sub-groups who benefit by HER-3 targeting agents.

Disclosure

All authors have declared no conflicts of interest.