Sir Peter MacCallum Department of Oncology - Theses
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ItemInvestigating the cell-of-origin of Serous Ovarian Tumours using single-cell transcriptomics( 2023-11)Serous ovarian cancer (SOC) is the deadliest type of female gynaecological tract cancer. Its cell of origin is currently debated. Finding the cell of origin of SOC could help with the development of novel diagnostic and screening biomarkers. I utilised single-cell transcriptomics bioinformatics methods to investigate cell types in normal human gynaecological tissues and serous ovarian tumours to investigate the cell of origin of serous ovarian cancer. Transcription profiles from single-nucleus and single-cell data generated from our lab and publicly available datasets of fresh human samples were evaluated in order to find and characterize epithelial cell populations in the premalignant and malignant states and derive their lineages. I identified novel rare progenitor cell populations in normal and premalignant fallopian tube and ovary. These stem cell populations expressed markers of stemness (ALDH1A3, ROR1, ROR2, PROCR) and serous ovarian cancer (CLDN1, WT1, MUC16). Since cancer is a tissue specific disease, I utilised these relevant normal tissue expression profiles to elucidate the identity of cell types in serous tumours based on the cell type identity preservation framework. Stromal cells previously annotated as cancer associated fibroblasts that were expressing mesenchymal and stemness markers were classified as part of the epithelial lineage in serous ovarian tumours. A differentiation trajectory indicated a linear epithelial lineage in all serous ovarian tumours irrespective of their grade. This lineage started from LGR5+ cancer stem cells progressing to ALDH1A3+ secretory progenitor cells, then to PAX8+ secretory cells and finally to FOXJ1+ ciliated cells. Functional evaluation of markers of precursor cell types indicated their quiescent and invasive properties and their possible role in angiogenesis. Here, I propose a stem cell model of serous ovarian tumour initiation and initial invasion.
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ItemDefining signalling pathways that control the response of endothelium to cancer therapy( 2018)Targeting non-transformed stromal components of the tumour microenvironment (TME) has become clinically attractive in treating cancer over the last few decades. On this basis, vascular endothelial growth factor A (VEGFA) inhibitors which suppress blood vessel sprouting (angiogenesis) by blocking VEGFA signalling have been developed and integrated into modern cancer treatment regimens. However, tumour response to VEGFA inhibitors is highly complex and variable. In addition to cancer cells, the TME is composed of various stromal cell types that play an important role in modifying the tumour response by, for example, supporting the development of resistance to VEGFA blockade. Moreover, the indiscriminate large-scale application of VEGFA inhibitors (with or without chemotherapeutic agents) in clinical oncology, resulting in overall modest patient benefit and the inevitable occurrence of resistance, has underscored a pressing need for rational use of these expensive agents. To address the challenges in deciphering the role of each TME component and thus the mechanisms of resistance, this Thesis focused on the main cellular target of VEGFA inhibitors — human microvascular blood endothelial cells (ECs). To identify molecular modifiers of the EC response to VEGFA inhibitors (in this Thesis bevacizumab, a humanised anti-VEGFA neutralising monoclonal antibody, was used), a pooled genetic screening platform was developed. This involved a three-dimensional microcarrier-based culture system, CRISPR–Cas9-driven genetic loss-of-function (LOF) and VEGFA-dependent serum-free culture conditions for applying selective pressure. A pooled kinome-wide CRISPR–Cas9-based screen identified 18 candidate genes that upon LOF were significantly enriched or depleted in the bevacizumab versus control treatment arm. Candidate evaluation using small interfering RNA (siRNA) validated ACTR2, BRD2, BRD3, BRD4, TAOK1 and TRRAP LOF as mediators of EC resistance to bevacizumab; TLK1 and TLK2 LOF as sensitisers of ECs to bevacizumab. Further analysis of the most significant validated candidate genes BRD2, BRD3 and BRD4 (encoding members of the bromodomain and extraterminal domain (BET) family of proteins) using the BET bromodomain inhibitors (BETi) JQ1 and I-BET762 reproduced the effect of siRNA-mediated knockdown of BRD2, BRD3, or BRD4 on the EC response to bevacizumab. Markedly, a survival- and/or proliferation-inhibiting effect of BETi was observed regardless of the presence of bevacizumab. However, this inhibitory effect was unexpectedly attenuated when cells were co-treated with bevacizumab under VEGFA-dependent culture conditions. These results collectively indicated an interaction between BETi and bevacizumab. Investigation of the mechanistic basis for such interaction using RNA sequencing suggested a role for epigenetic regulation of chromosomal activity in modifying the EC response to co-treatment with BETi and bevacizumab. With development and application of a minimally biased and systematic screening approach, this Thesis identified and validated novel molecular modifiers of the EC response to bevacizumab. A previously unreported interaction between BET protein activity and VEGFA signalling in the context of bevacizumab treatment in ECs was revealed. Importantly, these observations will prompt further investigation of the role of epigenetic regulation in vascular biology, tumour angiogenesis and response to cancer therapy. These findings could facilitate clinical development of predictive and/or response biomarkers and strategies to overcome therapeutic resistance, ultimately enabling the rational use of VEGFA inhibitors.