Dale, Katie; (2021) Functional identification and investigation of genes initiating chromosomal instability using CRISPR activation and high-throughput automated image analysis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Chromosomal instability (CIN), the dynamic state where cells experience increased structural and/or numerical chromosome segregation errors, is prevalent in cancer, where it contributes to aneuploidy and tumour evolution. Despite its profound consequences on human health, initiation of CIN in the early stages of tumourigenesis is not well understood. In this work multiple strategies were employed to investigate the effects of gene upregulation, as well as upregulation of the PIK3CA signalling pathway, a frequently altered pathway in many cancer types (Jamal-Hanjani et al. 2017; Teixeira et al. 2019). Combining CRISPR gene upregulation to model overexpression, high content imaging (HCS), and automated high-throughput image analysis, a pipeline was developed to screen for CIN and aneuploidy in a normal human cell line, RPE1. This provides a readout of micronuclei and centromere counts. Using this pipeline, upregulation of KIF11 was found to increase the proportion of cells exhibiting micronuclei, and cause significant deviation from the modal centromere count, indicating CIN and aneuploidy. Further investigation of this phenotype revealed that KIF11 upregulation causes spindle pole fragmentation, mitotic catastrophe, and chromosome congression defects. Centric and acentric lagging chromosomes were observed in cells that exhibited both normal and fragmented spindle poles. Mechanistically, KIF11 was shown to generate a force imbalance in the early stages, which could be partially rescued upon upregulation of HSET. MCF10A cell lines expressing PIK3CAH1047R at the endogenous genetic loci as a result of CRISPR genome editing were used to investigate the impact of increased signalling through the PIK3CA pathway on CIN, aneuploidy, and centrosome biology. This showed that PIK3CAH1047R increased the incidence of supernumerary centrosomes, and may play a role in structural CIN, but failed to identify any effect on numerical CIN. Finally, by chemically modulating PIK3CA activity, microtubule dynamics in response to PIK3CA pathway activation and inhibition were investigated.

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