Proximity-based assays for the detection of activated HER3, HER2/HER3 heterodimers and HER3/PI3K complexes in formalin-fixed, paraffin-embedded cell line controls and tumors.

Abstract

Abstract Abstract #4040 Although kinase-defective, the role of the HER3 receptor in driving tumor growth through dimerization with other HER family members is well recognized. There is increasing interest in developing therapeutic strategies to interfere with signaling through this receptor, either directly or indirectly. At the same time, there is recognition that the activation of HER3 could be an important biomarker of drug response and resistance. In particular the HER2/HER3 heterodimer has been extensively studied and, among all the possible homo and heterodimers formed by HER receptors, is considered the most mitogenic because of the ability of HER3 to effectively couple to the pro-survival PI3K/Akt pathway.
 Using the VeraTag™ technology, we have developed a suite of assays designed to detect quantitatively the activation status of the HER3 receptor by measuring HER3-specific phosphorylation and formation of HER3 signaling complexes in formalin-fixed, paraffin-embedded (FFPE) cell line controls and tissue samples.These assays are dual antibody-based and measure complexes of proteins in close association since signal is generated only when the antibodies are bound to target-specific epitope sites proximal to one another. A capillary electrophoresis instrument equipped with laser-induced fluorescence provides a highly sensitive and quantitative read-out of the assay. We report data on assays which detect a ligand-induced, 2-5-fold change in HER2/HER3 heterodimer formation, a 10-20 fold change in phosphorylated HER3 signal and as high as a 10 fold change in HER3/PI3K complex formation in a series of cell line controls, spanning a wide range of HER2 and HER3 receptor levels. FFPE data were verified with results from co-immunoprecipitation using antibodies identical to those used in the VeraTag assay. The influence of the HER2 and HER3 levels on the formation of HER2/HER3 heterodimers and correlation with HER3 phosphorylation and formation of the PI3K adaptor complex will be discussed.
 In addition, we have used these assays to landscape HER3 activation in a number of tissues, including breast and ovarian tumors, focusing particularly on HER2 positive breast cancer, where the HER2/HER3 heterodimer is thought to play a particularly significant role. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4040.