Abstract 2235: FRET assay to assess EGFR/HER2 dimerization in cancer cell lines
Authors: Waterhouse Benjamin R. , Gijsen Merel , Tullis Iain , Barber Paul , Vojnovic Boris , Kong Anthony 
Published: June 2012

Abstract
Expression level of the HER family is unreliable as a predictive marker for the outcome of targeted therapies in cancer. Only a third of HER2 positive breast cancer patients respond to trastuzumab monotherapy. Thus there is significant need for the development of new biomarkers and the role of dimerization may prove a more effective predictor than expression itself.
It has been shown that specific HER dimerization pairs are important. However, there has been no protocol proposed which can accurately assess dimerization to facilitate the development of an assay viable for use in a clinical setting. Previous attempts have either been based upon transfection of proteins whose interactions can be monitored which would not seem to be directly applicable to in-situ monitoring or do not explicitly separate true dimerization from mere colocalization. Our approach also allows intracellular localization to be visualized, this is significant as nuclear translocation of EGFR can lead to a number of signaling events.
Utilizing measurement of Foerster Resonance Energy Transfer (FRET) to assess distances of the order 7 nm between receptors we can quantify dimerization. Our secondary conjugation technique uses anti-mouse and anti-rabbit primary antibodies and species specific dye-conjugated secondary antibodies, with Alexa488 as the donor and Alexa546 as the acceptor dye. An increase in dimerization results in increased occurrence of FRET which can be measured as a reduction in donor fluorescence lifetime. We have validated our assay for dimerization of EGFR and HER2 in 3 cell lines, BT474, SKBr3 and A431, and furthermore in paraffin embedded cell pellets.
Comparison of preparations with donor antibodies alone with those containing donor and acceptor we demonstrate basal EGFR/HER2 dimerization levels are present (p&lt;0.025). This was enhanced by ligand EGF, producing further decrease in lifetime (p&lt;0.025). Whereas tyrosine kinase inhibitor gefitinib and HER3/HER4 ligand heregulin, which decreases EGFR/HER2 dimerization, showed an increase in lifetime. These results are consistent with documented changes seen using co-immunoprecipitation experiments.
We then applied the assay to monitor response to targeted therapies, the HER2 specific monoclonal antibodies, trastuzumab and pertuzumab. Trastuzumab is seen to decrease lifetime while dimerization inhibitor pertuzumab produces an increase. This is consistent with findings using biochemical methods.
Finally, we applied and validated our assay in paraffin embedded cell pellets. These data demonstrate the robustness of our assay and the potential clinical application to paraffin-embedded tissue microarrays in order to assess the contribution of HER signaling in-situ. The assay represents a proof of principle, from here this same technique could be applied to other dimerization pairs. This will allow the prognostic potential of dimerization to be assessed and used to inform treatment regimes.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2235. doi:10.1158/1538-7445.AM2011-2235