EGFR-HER2 Transactivation Viewed in Space and Time Through the Versatile Spectacles of Imaging Cytometry-Implications for Targeted Therapy.

Abstract

Ligand-induced formation of signaling platforms composed of homo- and/or heterodimers of receptor tyrosine kinases is considered essential for their activation and consequential contribution to the progression of many cancers. Epidermal Growth Factor Receptor (EGFR) acts as a signal receiver upon EGF binding and produces mitogenic input for many cells also through receptor-heterodimerization with its ligandless partner, Human Epidermal growth factor Receptor 2 (HER2). Ligand-driven transactivation is a key step leading to changes in the cell surface pattern of EGFR and HER2; their interaction plays a key role in various malignancies, especially when HER2 molecules are overexpressed. Our clinically relevant model system is the SK-BR-3 breast tumor cell line, overexpressing HER2 and moderately expressing EGFR. This cell line shows significant dependency on EGF-driven HER2 signaling. We studied changes in the interaction between EGFR and HER2 in the cell membrane upon EGF binding, applying various biophysical approaches with different time scales. Changes in molecular proximity were characterized by fluorescence lifetime imaging microscopy (FLIM) techniques assessing Förster resonance energy transfer (FRET), which confirmed the ligand-enhanced interaction of EGFR and HER2, followed by an increase in HER2 homoassociation. EGF binding and transactivation were reflected in the phosphorylation of both receptor types as well. At the same time, superresolution Airyscan microscopy and fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS), sensitive to changes in the size of stationary and diffusing aggregates, respectively, have revealed cyclic increases in the aggregation and stable co-diffusion of membrane-localized HER2, possibly caused by internalization and recycling, eventually leading to a new equilibrium. Such dynamic fluctuation of receptor interaction may open a window for the binding of therapeutic antibodies that are aimed at inhibiting heterodimerization, such as pertuzumab. The complementary array of state-of-the-art imaging cytometry approaches thus demonstrates a spatiotemporal pattern of spontaneous and induced receptor aggregation states that could provide mechanistic insights into the potential success of targeted therapies directed at the HER family of receptor tyrosine kinases.