Sir Peter MacCallum Department of Oncology - Theses
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ItemMechanisms regulating Drosophila neural stem cell proliferation( 2021)Background Neural stem cells are cells that can self-renew and differentiate to give rise to specialized cell types, such as neurons. The appropriate regulation of neural stem cell activities during development guarantees that the correct number of functional neurons are generated to form the adult nervous system. Dysregulation of this process can result in a number of neurological diseases. Too few neural stem cells (and their neuronal progeny) can lead to microcephaly, and too many neural stem cells (and their neuronal progeny) can lead to brain tumour formation. Therefore, understanding the mechanisms regulating neural stem cell activities may shed light on novel therapeutics for neurological diseases. Drosophila neural stem cells, the neuroblasts (NBs), have been an advantageous model for the study of neural stem cell biology during development and in disease contexts. In the developing larval Central Nervous System (CNS), NB activities are maintained by intrinsic regulatory machinery as well as extrinsic cues from glial cells, which form a microenvironment enwrapping NBs. Dysregulation of either mechanism can have severe consequences. Deregulation of NB intrinsic regulators such as a homeobox transcription factor called Prospero (Pros) can result in decreased differentiation and an expansion of NBs, resulting in increased brain size. The disruption of glial niche or glial derived signals can lead to an alteration of NB proliferation rate, thus affecting the number of neurons in the adult CNS. While multiple intrinsic factors regulating NB activities during development have been intensively studied, it remained poorly understood how NB intrinsic metabolic pathways are important for NB proliferation under physiological and disease context. Furthermore, the mechanisms of how NBs respond to extrinsic cues and how the glial niche relay extrinsic signals to control NB activities is still not well understood. Hence, the aim of my study is to expand our knowledge on how intrinsic metabolic adaptations and extrinsic cues regulate the progenitor pool of NBs and the rate at which neuronal progeny is produced. Aims, methods and results: In this work, I have addressed two key questions: 1) what metabolic rewiring events occur as a result of NB tumour formation caused by dedifferentiation? 2) How do glial niche and glial derived signals non-autonomously regulate NB proliferative activities? 1) Metabolic rewiring is known to accompany aberrant proliferation and has recently been shown to drive tumour growth. Utilising the Drosophila NB tumour model caused by deregulation of pros (a homeobox transcription factor), we conducted transcriptomics and metabolomics studies to examine metabolic changes that occur as a result of defective NB differentiation. I found that glutathione metabolism is altered as a result of pros loss-of-function (LOF) tumour formation. It is known that the Pros homologue PROX1 is deregulated in neuroblastoma. Therefore, findings from our work may inform the search of novel metabolite-based targeted therapies for the treatment of neuroblastoma. 2) In the larval CNS, NBs re-enter into cell cycle from a quiescent state in response to extrinsic signals from their microenvironment made up of glial cells. In my study, I have uncovered several new regulators of glia-NB interaction using genetic analyses. I found that lipid metabolism and Hedgehog (Hh) in the glial niche are involved in regulating NB proliferation. Glial Hh is autonomously required for the growth of glial niche, which is key in maintaining NB proliferation during development. Upon glial overgrowth mediated by FGF activation, excessive Hh from the glial niche triggers a delay in NB cell cycle progression. In this glial overgrowth context, Hh function is modulated by two lipid regulators, Fatty acid synthase 1 (Fasn1) and Lipid droplet storage 2 (Lsd-2). I demonstrated that Fasn1 can modulate Hh palmitoylation. However, it remains unclear how Lsd-2 modulates Hh function, despite the observation that Lsd-2 and Hh both localize to the surface of lipid droplets localized to glial cells. I also found that glial derived chitinase like proteins, called imaginal disc growth factors (IDGFs) are important non-autonomous regulators of NB proliferation. I showed that IDGF2 is necessary for NB exit from quiescence and is required to sustain NB proliferation at later larval stages. Collectively, these findings revealed novel mechanism by which Drosophila neural stem cell activities are regulated during development and during tumorigenesis.
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ItemTransdifferentiation of cancer stem cells in brain tumours: Lessons from Drosophila neural stem cells( 2020)Background: In human brain cancers, glioblastoma stem cells (GSCs) originate from neoplastic transformation of neural stem cells (NSCs) or dedifferentiation of other neural cells. Similar to normal NSCs, GSCs possess stem-cell properties to self-renew and differentiate into multiple neural lineages. However, GSCs are more plastic than normal NSCs, as they can transdifferentiate into other cell types. At the present, we do not fully understand the cellular step-wise conversion from GSCs into other distinct cell lineages, and the molecular mechanism responsible for this event. Drosophila NSCs, called neuroblasts (NBs), also asymmetrically divide to renew themselves, and generate neurons or glia that make up the adult central nervous system. Disrupting either asymmetric cell division or neuronal maintenance allows differentiated cells to dedifferentiate into ectopic NBs, which then continue to proliferate and form tumours. Methods and aims: We utilised several in vivo brain tumour models in Drosophila to study how GSCs function in brain cancers and how they undergo transdifferentiation. In this thesis, I induced loss-of-function of transcription factors Prospero or Nerfin-1 in NB lineages to generate dedifferentiation-driven tumours, and found a class of cells which exhibited glial cell identity. I sought to investigate the behaviours and characteristics of these ectopic glia to elucidate some aspects about GSC transdifferentiation by answering three questions: (1) What are the cell numbers and cell types within the pros- and nerfin-1- tumours. (2) Do ectopic glia arise by transdifferentiation of NBs in the tumours? (3) Which transcription factors and signal transduction pathways drive the formation of ectopic glia, and are ectopic glia required for tumour growth? Results: (1) I found that the expansion of ectopic glia population is correlated with overall tumour growth. Ectopic glia exhibit glial identity and their formation is not dependent on the location of tumours in the central nervous system. (2) By performing live-cell imaging of pros- tumours and molecular marker analysis, I found a subset of NBs switch to glial cell fate. (3) I performed genetic experiments to manipulate the transcription factors dichaete, tailless and glial cells missing (gcm) in pros- tumour and found that they are required for ectopic glial formation and tumour growth. Their target gene reversed polarity (repo) regulates the formation of ectopic glia, which in turn, promote the tumour growth. I showed that Notch promotes tumour growth independently of its effect on ectopic glial formation, as Notch regulates the tumour growth in the absence of ectopic glia. I also showed that FGF signalling pathway promotes tumour growth by regulating ectopic glia formation, as it does not affect the tumour growth in the absence of ectopic glia. Hippo pathway also plays a role in promoting the formation of ectopic glia and tumour growth. Our study of pros- and nerfin-1- tumour models in the context of transdifferentiation may extend our understanding of the biology of NBs and may shed light on GSC behaviours upon their transdifferentiation into different cell types. We can use the underlying mechanisms of these phenotypes to gain a better understanding of the transdifferentiation events at the molecular and cellular levels. As most genes and signalling pathways examined in this study are also found in human brain cancers, this study will enhance the knowledge of how cell fate changes can influence the tumour malignancy.
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ItemCooperative tumourigenesis : analysis of novel tumour suppressors in ras oncogene driven epithelial tumours( 2015)Cancer is a cooperative process, involving mutations in multiple genes. Activation of a cancer-driving gene, the Ras small GTPase, via a mutation that locks Ras in the GTP-bound active form (RasV12), occurs in ~30% of human cancers. However, alone it is not sufficient for tumour formation. A loss of function screen previously performed in the vinegar fly, Drosophila melanogaster, identified 947 genes that potentiate RasV12-mediated tumourigenesis and metastasis (Zoranovic, et al. in prep.). This list has been narrowed down to 234 genes that 1) show increased tumourigenicity with RasV12 in vivo, 2) are in the top 100 genes down-regulated in human cancer, and 3) are known to regulate the cytoskeleton, polarity, adhesion or cell motility. This study has successfully confirmed involvement of autophagy-related genes Atg8a, Atg7 in regulating RasV12-mediated proliferation in the Drosophila eye epithelial tissue using the UAS/GAL4 system. The study identified the autophagy-related genes Atg1, Atg3, Atg4, Atg5, Atg6, Atg7, Atg8a, Atg12 and Atg101 that when knocked down cooperate with RasV12 and lead to increased tissue overgrowth in the Drosophila eye epithelium. Atg8a was chosen as the representative target gene to investigate this cooperation. It was observed that Atg8a cooperates with RasV12 through the Raf pathway. The role of p62 in this Ras-mediated cooperation with Atg8a was also examined and it was found that p62 levels increase in RasV12+ Atg8aRNAi expressing tissue in comparison with control. Investigations were also carried out to ascertain if knockdown of Atg genes cooperate with Ras through the JNK pathway. It was discovered that in the presence of oncogenic Ras, knock down of Atg8a increases the expression of the JNK target MMP1. The finding of this work could lead to use of this autophagy related genes as prognostic markers in Ras-driven oncogenesis and might reveal effective therapeutic targets to combat this deadly disease.