Single-Particle Tracking of the Human Epidermal Growth Factor Receptors (HER) 1-3 in a Breast Cancer Model: the Effect of Tyrosine Kinase Inhibitors

Abstract

Over-expression and mutational activation of the Human Epidermal Growth Factor Receptor (HER) 1-4 receptor family are implicated in tumorigenesis and cancer progression. Perturbations of their associated signalling cascades and feed-back loops can amplify aberrant signalling. Inhibition of these signals is a central objective of anti-cancer targeted therapeutics, but to date most drugs have limited success. The reasons are not understood and underscore a lack of understanding of the molecular basis of receptor signalling and drug action. At the heart of these processes are combinatorial interactions leading to receptor homo- and hetero-dimerization To characterise the interactions between HER family homo- and hetero-dimers in basal and activated states, receptors were labelled in their non-active configuration with Affibodies conjugated to fluorophores or activated by fluorescently-tagged ligands. This will enable future application of the method to primary samples. Cell culture substrates and fluorescent dyes were optimised to reduce the incidence of non-specific probe adhesion and a set of parameters to guide the choice of fluorescent dyes for Single-Molecule applications was determined. Alexa Fluor 488- and CF640R- tagged probes were used for pairwise live-cell tracking of HER1-3 receptors in presence or absence of clinical-grade tyrosine kinase inhibitors. These experiments highlighted the role of tyrosine kinase activity in determining HER1 diffusion at the plasma membrane and likelihood of interaction. Activated HER1 homo-interactions and hetero-interactions with HER2 were the predominant colocalisation pairs identified. Precise determination of the dwell-time distribution of all pairs is pending and will require more precise assessment of photobleaching lifetime contaminations and denoising. Analysis of the diffusion of HER1 with a novel, globally-optimised Bayesian tracking algorithm developed by Dr Wareham (University of Cambridge) revealed the presence of two populations of receptors characterised by their directional motion components. The most directional population depends on the presence of an intact actin cytoskeleton and on tyrosine kinase activity. The methods presented in this thesis, will be extended to study the dynamics and kinetics of the whole HER family, laying the foundations for the construction of a quantitative model of the system.

Date of Award	2014
Original language	English
Awarding Institution	King's College London
Supervisor	Tony Ng (Supervisor)