Anton, KA; (2013) Molecular analysis of interactions between normal and transformed epithelial cells at early stages of cancerogenesis. Doctoral thesis , UCL (University College London).

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Abstract

Carcinomas begin with a single transformed cell in an otherwise normal epithelial monolayer. To study the nature of interactions between a transformed cell and its neighbours at this initial stage of tumourigenesis a tetracycline-inducible system driving expression of a constitutively active form of oncogenic Ras (RasV12) was established in MDCK epithelial cells by the Fujita laboratory. Upon interaction with normal cells, RasV12 cells most commonly undergo apical extrusion from an epithelial monolayer. In order to identify proteins and pathways involved in interactions between normal and transformed cells, I have performed several biochemical screens described in this thesis. Firstly, I have shown that Hsp90β, identified previously in a 2D gel screen, is increased in RasV12 cells surrounded by normal neighbours in a non-cell-autonomous fashion. By using inhibitors and a dominant negative form of Hsp90, I have shown that upregulation of this chaperone is most likely a part of a stress response delaying extrusion of transformed cells. Secondly, I have performed a screen for tyrosine phosphorylated proteins and identified myosin IE and plectin as modified in mixed cultures of normal and transformed cells. Finally, I have undertaken quantitative mass spectrometry of phosphorylated peptides using SILAC labelling to assess changes in RasV12 cells upon their interaction with normal cells. In this screen I have found that an actin anticapping protein, VASP, is phosphorylated on serine 239 in RasV12 cells interacting with normal cells. This modification is known to inhibits its related to actin function. I have shown that depletion of VASP in RasV12 cells results in their enhanced extrusion from normal monolayers, most likely due to their compromised attachment. The phosphorylation on serine 239 may be an early step in extrusion, contributing to disassembly of focal adhesions and stress fibres in transformed cells. I have also studied the role of another protein identified in the SILAC screen, MRCKβ, and shown that its depletion results in enhanced extrusion of RasV12 cells from normal monolayers.

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