Abstract
The expression level of the HER family is unreliable as a predictive marker for targeted therapies in cancer. Thus, there is a need to develop other biomarkers, which can be used to accurately select responsive patients for targeted therapies. The HER dimerization status may be more important than HER receptor expression per se in determining sensitivity or resistance to a given therapeutic agent. The aim of the study is to develop a FRET assay using dye conjugated secondary antibodies to assess HER receptor dimerization. Using primary antibodies from different species in conjunction with Alexa488 and Alexa546 conjugated secondary antibodies, we validated our EGFR/HER2 dimerization assay in three cell lines, EGFR positive A431 cells as well as HER2 positive breast cell lines BT474 and SKBR3 cells. Finally, we applied our assay to assess EGFR/HER2 dimerization in paraffin embedded cell pellets. Our results show promise for the assay to be applied to tumor samples in order to assess the prognostic significance and predictive value of HER receptor dimerization in various cancers.
Highlights
Dysregulation of HER (ErbB) receptors has been implicated in several cancers
Using primary antibodies for EGFR and HER2 from two different species in conjunction with speciesspecific secondary antibodies conjugated to Alexa488 or Alexa546, we first assessed the dimerization of EGFR and HER2 in fixed cell samples
It was hypothesized that upon ligand simulation, there would be an increase in EGFR/HER2 dimerization and Förster Resonance Energy Transfer (FRET) between the two conjugated flurophores, The overlap in the donor (Alexa488) emission spectrum and acceptor (Alexa546) absorption spectrum allows FRET to occur, resulting in a decrease of donor fluorescence lifetime, indicative of EGFR/HER2 dimerization (Figure 1)
Summary
Dysregulation of HER (ErbB) receptors has been implicated in several cancers. For example, EGFR is frequently over-expressed in Head and Neck Squamous Cell Carcinoma (HNSCC) and is correlated with poor disease-free survival and overall survival in these patients [1, 2]. Such mutations are uncommon in other cancers like HNSCC and may not be predictive of sensitivity to EGFR inhibitors in these cancers [11] For those cancers without EGFR mutations, it has been shown that the expression levels of EGFR do not predict the success of these drugs and the response rate to these inhibitors remains poor [9, 12]. One reason for this is that the HER receptors are able to form alternative dimers and can compensate the loss of function of one receptor during targeted therapies [13, 14]. The ability to assess the dimerization pairs within tumours could be useful as a prognostic or predictive biomarker for targeted therapies in cancer
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