Critical roles for EGFR and EGFR-HER2 clusters in EGF binding of SW620 human carcinoma cells

TY - JOUR

T1 - Critical roles for EGFR and EGFR-HER2 clusters in EGF binding of SW620 human carcinoma cells

AU - Wollman, Adam J M

AU - Fournier, Charlotte

AU - Llorente-Garcia, Isabel

AU - Harriman, Oliver

AU - Payne-Dwyer, Alex L

AU - Shashkova, Sviatlana

AU - Zhou, Peng

AU - Liu, Ta-Chun

AU - Ouaret, Djamila

AU - Wilding, Jenny

AU - Kusumi, Akihiro

AU - Bodmer, Walter

AU - Leake, Mark C

N1 - © 2022 The Authors

PY - 2022/5/25

Y1 - 2022/5/25

N2 - Epidermal growth factor (EGF) signalling regulates normal epithelial and other cell growth, with EGF receptor (EGFR) overexpression reported in many cancers. However, the role of EGFR clusters in cancer and their dependence on EGF binding is unclear. We present novel single-molecule total internal reflection fluorescence microscopy of (i) EGF and EGFR in living cancer cells, (ii) the action of anti-cancer drugs that separately target EGFR and human EGFR2 (HER2) on these cells and (iii) EGFR-HER2 interactions. We selected human epithelial SW620 carcinoma cells for their low level of native EGFR expression, for stable transfection with fluorescent protein labelled EGFR, and imaged these using single-molecule localization microscopy to quantify receptor architectures and dynamics upon EGF binding. Prior to EGF binding, we observe pre-formed EGFR clusters. Unexpectedly, clusters likely contain both EGFR and HER2, consistent with co-diffusion of EGFR and HER2 observed in a different model CHO-K1 cell line, whose stoichiometry increases following EGF binding. We observe a mean EGFR : EGF stoichiometry of approximately 4 : 1 for plasma membrane-colocalized EGFR-EGF that we can explain using novel time-dependent kinetics modelling, indicating preferential ligand binding to monomers. Our results may inform future cancer drug developments.

AB - Epidermal growth factor (EGF) signalling regulates normal epithelial and other cell growth, with EGF receptor (EGFR) overexpression reported in many cancers. However, the role of EGFR clusters in cancer and their dependence on EGF binding is unclear. We present novel single-molecule total internal reflection fluorescence microscopy of (i) EGF and EGFR in living cancer cells, (ii) the action of anti-cancer drugs that separately target EGFR and human EGFR2 (HER2) on these cells and (iii) EGFR-HER2 interactions. We selected human epithelial SW620 carcinoma cells for their low level of native EGFR expression, for stable transfection with fluorescent protein labelled EGFR, and imaged these using single-molecule localization microscopy to quantify receptor architectures and dynamics upon EGF binding. Prior to EGF binding, we observe pre-formed EGFR clusters. Unexpectedly, clusters likely contain both EGFR and HER2, consistent with co-diffusion of EGFR and HER2 observed in a different model CHO-K1 cell line, whose stoichiometry increases following EGF binding. We observe a mean EGFR : EGF stoichiometry of approximately 4 : 1 for plasma membrane-colocalized EGFR-EGF that we can explain using novel time-dependent kinetics modelling, indicating preferential ligand binding to monomers. Our results may inform future cancer drug developments.

KW - Carcinoma/metabolism

KW - Cell Line, Tumor

KW - Colonic Neoplasms/metabolism

KW - Epidermal Growth Factor/metabolism

KW - ErbB Receptors/metabolism

KW - Humans

KW - Phosphorylation

KW - Receptor, ErbB-2/metabolism

KW - Signal Transduction

U2 - 10.1098/rsif.2022.0088

DO - 10.1098/rsif.2022.0088

M3 - Article

C2 - 35612280

SN - 1742-5689

VL - 19

JO - Journal of the Royal Society Interface

JF - Journal of the Royal Society Interface

IS - 190

M1 - 20220088

ER -