Investigating the origins of polyploidy in Barretts oesophagus and oesophageal adenocarcinoma

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Scott, Stacey 

Investigating the origin(s) of polyploidy in Barrett’s oesophagus and Oesophageal adenocarcinoma. Oesophageal adenocarcinoma (OEAC) is preceded by the condition Barrett’s oesophagus (BE), in which the stratified squamous mucosa of the oesophagus becomes damaged from exposure to stomach acid, resulting in a transformation into a simple columnar mucosa typical of that of the small intestine. A key step in the progression from BE to OEAC is the acquisition of polyploidy after p53 loss via an unknown genome doubling event. This increase in genomic material is likely to contribute to OEAC evolution and diversification. To identify the origin of polyploidy in OEAC, I analysed mitosis in both BE and OEAC cell lines by time-lapse imaging and immunofluorescence (IF) and found that about 12% of dividing OEAC cells present with an abnormal mitotic phenotype, the most prevalent being a defect in chromosome congression and alignment. Whole genome and RNA sequence analyses of OEAC cell lines indicated numerous copy number changes and gene expression alterations of important inner and outer kinetochore components including genes within the CCAN and KMN networks. These findings were confirmed by the analysis and comparison of the expression and localisation of KMN proteins in OEAC vs. BE cells by IF and quantitative mass spectrometry analysis of immunoprecipitations of KMN components. Finally, I also observed similar chromosome congression defects in OEAC organoids. In sum, my results suggest that subtle changes in the levels of kinetochore proteins are the cause of chromosome congression defects in OEAC cells, which in turn trigger mitotic slippage and polyploidy in OEAC cells.
Identification of factors involved in crosstalk between actin filaments and microtubules. The eukaryotic cytoskeleton is vital in the control of a number of cellular processes including cell division, maintaining cell shape, establishment of cell polarity, cell motility and cell function. The dynamicity of the cytoskeleton is mediated by actin filaments and microtubules; whilst these structures control different aspects of the cellular behaviour, a cross talk must exist between the two in order to orchestrate a response to stimuli. It is already known that an actin-microtubule cross talk exists however there is little understanding about the mechanisms and signalling pathways that control this process. Experimental evidence indicates that the addition of actin depolymerising drug Latrunculin-A to cultured Drosophila cells results in a rapid change to cell shape, characterised by the formation of long microtubule filled protrusions. RNAi screening of Drosophila kinesins revealed a potential role for kinesin heavy chain (KHC), Klp10A and Klp61F in bundle formation; knockdown of these kinesins resulting in the formation of much shorter microtubule bundles compared to controls. As work was being carried out on this screen a study by Jolly et al., (2016) was published outlining a similar line of investigation, in which they had also identified KHC as being involved in microtubule bundle formation following actin depolymerisation. This raised concern regarding the progression of the project and hence the decision was made to cease any further investigation.

D'Avino, Pier Paolo
Polyploidy, Barretts oesophagus, Oesophageal adenocarcinoma, Mitosis
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
MRC (1644735)
MRC (1644735)