RhoA Signalling in Ovarian Cancer

UoM administered thesis: Phd

  • Authors:
  • Harry Warner

Abstract

Epithelial Ovarian Cancer is the 6th most common cancer affecting women worldwide. Despite decades of research, the survival rate for epithelial ovarian cancer has barely changed. Therefore the development of new treatments is essential. As with most epithelial derived cancers, the pathology of epithelial ovarian cancer is associated with the metastatic migration of cancer cells from the primary tumour to secondary sites. In this PhD I utilise mass spectrometry based proteomics to characterise the Rho-family GTPase RhoA. RhoA signalling is of central importance to cell migration in 3D environments, both in vitro and in vivo. Furthermore, RhoA signalling has been shown to drive the formation of invasive filopodia in response to the upregulated endocytic recycling of the α5β1 integrin, in epithelial ovarian cancer cells. The role of RhoA signalling in forming invasive filopodia is thought to be critical in vivo, as the overexpression of the α5β1 integrin is correlated with a poor prognostic outcome in patients. Therefore this PhD focussed on identifying both general RhoA interactors as well as RhoA interactors that drive invasive migration in response to the up-regulated endocytic recycling of the α5β1 integrin. In order to study RhoA signalling in epithelial ovarian cancer cells a methodology was developed to purify RhoA interactors. Central to this methodology was the GFP-trap protocol, which allows for the purification of GFP-tagged proteins. Following the development of this methodology, a large scale experiment was carried out to identify RhoA interactors that were sensitive to the up-regulated endocytic recycling of the α5β1 integrin. The dataset was then subject to extensive bioinformatics analysis to provide novel insight into RhoA signalling. This extensive analysis allowed for the prioritisation of novel RhoA interactors for further study, through the use microscopy based techniques. Specifically the biosynthetic traffic trafficking chaperones Surf4 and TMED10 were selected, as their associated with RhoA is cRGDfV sensitive. In order to study Surf4 and TMED10, their expression was supressed through the use of siRNA. Various microscopy-based assays were then used to determine how the loss of Surf4 and TMED10 influences RhoA signalling and cell migration. In summary I have identified numerous novel RhoA interactors, a number which may drive epithelial ovarian cancer metastasis.

Details

Original languageEnglish
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Supervisors/Advisors
Award date1 Aug 2019