Muliaditan, D. | Search bioRxiv

bioRxiv2019

Replication Stress Generates Multiple Distinct Classes of Copy Number Alterations

BackgroundWe previously showed that a major driver of cancer chromosomal instability (CIN) is replication stress, the slowing or stalling of DNA replication. However, the precise drivers of replication stress in cancer and the mechanisms by which these cause CIN and influence tumour evolution remain unclear. Common fragile sites are well-known genomic locations of breakage after aphidicolin-induced replication stress, but their precise causes of fragility are debated, and additional genomic consequences of replication stress are not fully explored. ResultsUsing single cell sequencing we detected DNA copy number alterations (CNAs) caused by one cell cycle under replication stress in diploid non-transformed cells. Aphidicolin-induced replication stress caused multiple types of CNAs associated with different genomic regions and features. Coupling cell type-specific analysis of CNAs to gene expression and single cell replication timing analyses allowed us to pinpoint the causative large genes of the most recurrent chromosome-scale CNAs. In RPE1 cells these were largely confined to three sites on chromosomes 1, 2 and 7 and generated acentric lagging chromatin and micronuclei containing these chromosomes. Different replicative stresses generated distinct profiles of CNAs providing the potential to interpret specific replication stress mechanisms from cancer cells. ConclusionsChromosomal instability driven by replication stress occurs via focal CNAs and chromosome arm-scale changes, with the latter confined to a very small subset of chromosome regions, potentially heavily skewing cancer genome evolution trajectories. Single cell CNA analysis thus reveals new insights into the impact of replication stress on the genome and provides a platform to further dissect molecular mechanisms involved in the replication stress response and to gain insights into how replication stress fuels chromosomal instability in cancer.

molecular biology↗

bioRxiv2019

Mechanisms of Chromosomal Instability in High-grade Serous Ovarian Carcinoma.

Chromosomal instability (CIN), the continual gain and loss of chromosomes or parts of chromosomes, occurs in the majority of cancers and confers poor prognosis. Mechanisms driving CIN remain unknown in most cancer types due to a scarcity of functional studies. High-grade serous ovarian carcinoma (HGSC), the most common subtype of ovarian cancer, is the major cause of death due to gynaecological malignancy in the Western world with chemotherapy resistance developing in almost all patients. HGSC exhibits high rates of chromosome aberrations and knowledge of causative mechanisms is likely to represent an important step towards combating the poor prognosis of this disease. However, very little is known about the nature of chromosomal instability exhibited by this cancer type in particular due to a historical lack of appropriate cell line models. Here we perform the first in-depth functional characterisation of mechanisms driving CIN in HGSC by analysing eight cell lines that accurately recapitulate HGSC genetics as defined by recent studies. We show, using a range of established functional CIN assays combined with live cell imaging and single molecule DNA fibre analysis, that multiple mechanisms co-exist to drive CIN in HGSC. These include supernumerary centrosomes, elevated microtubule dynamics and DNA replication stress. By contrast, the spindle assembly checkpoint was intact. These findings are relevant for developing therapeutic approaches to manipulating CIN in ovarian cancer, and suggests that such approaches may need to be multimodal to combat multiple co-existing CIN drivers.

cancer biology↗