Pinner, S.; (2008) Mechanisms of cancer cell motility in vivo. Doctoral thesis , University of London.

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Abstract

This thesis describes investigations into mechanisms responsible for cancer cell motility in vivo. Chapters 1 and 2 provide a review of current literature in this field and also describe the techniques used to generate the following the results. Chapter 3 describes a candidate-based approach to investigate whether ROCK1 might be regulated by phosphorylation. Mutagenesis of ROCK1 was carried out at 3 chosen sites (T233 T380 T398) in the activation loop and the hydrophobic domain and the phenotypes of the mutants were analysed. Chapter 4 describes a parallel approach finding phosphorylation sites in ROCK1 by mass spectrometry. From these results T518 was chosen for further investigation and its possible function is investigated. Chapter 5 describes an siRNA screen designed to identify novel regulators of the cortical acto-myosin cytoskeleton. The read-out for this was based on the disruption of rounded blebbing morphology of A375 cells cultured on 3D gel matrices. The rounded morphology is similar to that observed in amoeboid cancer cell motility in vivo, therefore we hypothesised that genes required for contracted, rounded morphology might also be required for motility. Results identified PDK1 amongst other genes as a potential regulator of contractile forces in A375 cells and the role of PDK1 was investigated further. It was found that PDK1 was required both in vitro and in vivo for amoeboid cell motility. Chapters 6,7 and 8 detail the investigations into the mechanism of how PDK1 regulates the cytoskeleton and amoeboid cell motility. It was shown that PDK1 was responsible for the localisation of ROCK1 but not ROCK2 at the plasma membrane. This regulation was achieved by the direct binding of ROCK1 to PDK1. It was further found that PDK1 was able to compete with and prevent RhoE, a negative regulator of ROCK1, from binding. Chapter 9 investigates the relationship between cell morphology, motility and pigment production. It was found that it was possible to image melanin containing vesicles using multiphoton excitation, and using this technique, the motile behaviour of pigmented melanoma cells was observed in vivo. It was found that motile invasive cells tended to contain less melanin than non-motile cells suggesting that they were less well differentiated. This chapter details investigations into what differences in signalling could be responsible for a switch to a de-differentiated, more invasive/metastatic phenotype. The final chapter discusses the findings contained within this thesis and the possible implications.

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