Unconjugated
Epidermal growth factor receptor (EGFR) signalling is activated by ligand-induced receptor dimerization. Notably, ligand binding also induces EGFR oligomerization, but the structures and functions of the oligomers are poorly understood. Here, we use fluorophore localization imaging with photobleaching to probe the structure of EGFR oligomers. We find that at physiological epidermal growth factor (EGF) concentrations, EGFR assembles into oligomers, as indicated by pairwise distances of receptor-bound fluorophore-conjugated EGF ligands. The pairwise ligand distances correspond well with the predictions of our structural model of the oligomers constructed from molecular dynamics simulations. The model suggests that oligomerization is mediated extracellularly by unoccupied ligand-binding sites and that oligomerization organizes kinase-active dimers in ways optimal for auto-phosphorylation in trans between neighbouring dimers. We argue that ligand-induced oligomerization is essential to the regulation of EGFR signalling.
Introduction: Although oestrogen is essential for the development of the normal breast, adult mammary stem cells are known to be oestrogen receptor alpha (ER) negative and rely on paracrine signals in the mammary epithelium for mediation of developmental cues. However, little is known about how systemic oestrogen regulates breast cancer stem cell (CSC) activity.
Methods: Here, we tested the effects of oestrogen on CSC activity in vitro and in vivo and investigated which paracrine signalling pathways locally mediate oestrogen effects.
Results: CSC-enriched populations (ESA+CD44+CD24low) sorted from ER positive patient derived and established cell lines have low or absent ER expression. However, oestrogen stimulated CSC activity demonstrated by increased mammosphere and holoclone formation in vitro and tumour formation in vivo. This effect was abrogated by the anti-oestrogen tamoxifen or ER siRNA. These data suggest that the oestrogen response is mediated through paracrine signalling from non-CSCs to CSCs. We have, therefore, investigated both epidermal growth factor (EGF) and Notch receptor signals downstream of oestrogen. We demonstrate that gefitinib (epidermal growth factor receptor (EGFR) inhibitor) and gamma secretase inhibitors (Notch inhibitor) block oestrogen-induced CSC activity in vitro and in vivo but GSIs more efficiently reduce CSC frequency.
Conclusions: These data establish that EGF and Notch receptor signalling pathways operate downstream of oestrogen in the regulation of ER negative CSCs.