Modelling chromosomal instability in high-grade serous ovarian cancer

Abstract

High Grade Serous Ovarian Cancer (HGSOC) originates in the fallopian tube and is characterized by near-ubiquitous TP53 mutations. High levels of chromosomal instability (CIN) and resulting chromosomal copy number changes (aneuploidy) are another defining feature. Consequences of CIN in cancer cells are intratumoural genetic heterogeneity, cancer genome evolution, therapy resistance and a propensity for metastasis. CIN describes the perpetual mis-segregation of chromosomes as a result from errors in mitosis or interphase DNA replication stress. Since mutations in mitosis and DNA replication controlling genes are rare in HGSOC, I aimed to assess if genetic aberrations characteristic of HGSOC, specifically in TP53, BRCA1 and MYC, are sufficient to induce CIN. For this purpose, I first validated the human, fallopian tube-derived, non-transformed, hTERT-immortalized cell line FNE1 as a useful model system. Importantly, FNE1 cells are p53-proficient, chromosomally stable and near-diploid. Subsequently, I mutagenized the tumour suppressor genes TP53 and BRCA1 and overexpressed the oncogene MYC using CRISPR/Cas9 in combination with lentiviral vectors. This led to the establishment of TP53 single-, TP53/BRCA1 and TP53/MYC double- and TP53/BRCA1/MYC triple-mutant FNE1 subclones. Mutant FNE1 cells were then subjected to analyses of their genome by multiplex interphase Fluorescence in situ Hybridization (miFISH) and single-cell, shallow depth whole genome sequencing (scWGS). Analyses of two TP53/BRCA1/MYC triple-mutant FNE1 sub-clones revealed the emergence of tetraploidy and diverse gains and losses in comparison to wild-type FNE1 cells which suggests on-going CIN. Strikingly, an increase in aneuploidy could already be observed in TP53 single-mutant cells. To elucidate potential mechanisms causing CIN in the mutant subclones, RNA sequencing was performed. Gene set variation analysis (GSVA) revealed increased enrichment scores of Mitotic spindle, G2/M checkpoint, E2F targets and DNA replication gene sets in TP53 single-mutant cells alone suggesting early cell cycle deregulation as a result of p53 loss. Indeed, the G2/M checkpoint and E2F target gene sets were further enriched in TP53/BRCA1 and TP53/MYC double- and TP53/BRCA1/MYC triple-mutant FNE1 subclones. Taken together, these data suggest transcriptional rewiring of the cell cycle upon p53- loss, which is exacerbated by additional BRCA1 perturbation and MYC overexpression, as a cause of CIN in HGSOC.

Date of Award	1 Aug 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	David Gilham (Supervisor) & Stephen Taylor (Supervisor)