Clonal events leading up to cancer initiation
Rather than just a simple accumulation of mutations, cancer formation can be thought of as a Darwinian process, where evolutionary pressures select for cellular clones that expand and eventually turn cancerous. Many cancers are the consequence of infectious diseases (e.g., cervical cancer, nasopharyngeal cancer, liver cancer) or environmental toxins (e.g., asbestos or UV light). In this project, we will study clonal events during cancer initiation using hepatitis B virus integration into host DNA as a model. This is thought to induce either ER stress (driving a pro-carcinogenic phenotype) or immune tolerance, resulting in cancer formation and reduced immune surveillance. This project will tease out the particular roles of DNA integration events in cancer formation.
Our previous studies showed that clonal cell proliferation prior to tumour formation is independent of the site of virus integration. Here, we focus on the identity of, transcription from, and mechanisms that result in virus integration into host DNA. We will use the following independent, but complementary approaches:
- An in vitro model of integration combined with CRISPR-Cas9 gene editing. Here, we place integrations at specific cellular sites after infection with patient blood, allowing us to characterise the complete integrated sequences.
- An in vivo model of integration. Here, we will observe how virus integration alters the growth of cells in its native liver microenvironment.
- A high-throughput screening assay with a “reporter” virus. Here, we will undertake genome-wide screening (e.g. siRNA, CRISPR knock-out, cDNA library, etc.) of cellular targets to determine if the expression of certain genes increase the rate of integration.
Students working on this project can expect to learn the following techniques: CRISPR-Cas9-mediated gene editing, single-cell PCR, state-of-the-art microscopy, mathematical modelling, bioinformatics analysis, cell culture (including virus production), animal handling, flow cytometry, and/or high-throughput screening assays.