Insights into the mechanisms of HGF-mediated cell migration.
Doctoral thesis, UCL (University College London).
c-Met (the Hepatocyte Growth Factor Receptor) is a receptor tyrosine kinase, implicated in various human tumours. Ligand-induced c-Met activation and internalisation was described to play a role in Extracellular signal Regulated Kinase (ERK) phosphorylation and plasma membrane accumulation, with consequent effects on migratory responses. Initial work in this thesis illustrated that this accumulation was prevented using pharmacological agents to disrupt the microtubule network, and more specifically the microtubule motor proteins. To find specific players in this process, RNA interference was used as a tool to silence gene expression, and a high throughput HGF-mediated wound-healing assay was used as a readout. An siRNA library encoding more than 1,400 human genes targeting kinases, phosphatases, motor proteins and genes predicted to influence cell motility, was screened in a lung carcinoma cell line, A549. From a collection of hits identified in the primary screen, 100 were chosen for further validation. These hits fell into three categories: those that had a general effect on motility (but not having any effect on HGF-inducibility), those that markedly reduced the HGF-induced motility and those that increased the effect of HGF on motility. Twenty validated hits were selected for more extensive analysis. Four additional oligos were tested individually and as pools to add to the selection process. The top twelve highly validated hits were selected for more thorough investigation, including effects on cell speed, c-Met expression downregulation, localisation, and signalling. In addition, connection between hits and ERK subcellular localisation was also investigated. The screening approach was validated by the finding that two known players of the HGF-provoked response were identified, namely c-Met and ERK2. Although additional hits do not define the machinery needed to move ERK within cells, they do provide an insight into processes behind HGF-induced cell migration.
|Title:||Insights into the mechanisms of HGF-mediated cell migration|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Medical Sciences > Medicine (Division of) > Wolfson Inst for Biomedical Research|
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