Pathology - Theses
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ItemFunctional genomics analysis of interplay between cell polarity and oncogenic RAS-MAPK signalling( 2014)The development of cancer is a complex process and progression towards malignancy requires the cooperation of multiple transformation events. This often involves mutations or aberrant expression of various oncogenes and tumour suppressors. Whilst epithelial tumours are inherently heterogeneous, and each tumour type and sub-type is molecularly distinct, one of their pervasive features is disruption to normal epithelial cell polarity and tissue architecture. Indeed it has now emerged that the polarization and differentiation of epithelial cells is innately tumour suppressive, and deregulation of epithelial polarity often correlates with invasive and metastatic disease. Conversely, evidence from experimental model systems indicates that the enforced expression of core polarity proteins such as Scribble is sufficient to potently suppress cellular transformation driven by oncogenes such as RAS. Despite an indisputable link between deregulation of the polarity network and the process of oncogenic transformation, little is known about how this network functions collectively on a systems-level within human epithelial cells, nor how engagement of this network may mediate innate mechanisms of tumour suppression. Here I have undertaken a detailed systems-level analysis to identify and characterize the interface between the global epithelial polarity network and oncogenic RAS-MAPK signalling in both normal and malignant human epithelial cells. This was achieved through a genome-scale RNAi functional genomics screen carried out in human epithelial cells coexpressing both oncogenic RAS and the polarity protein Scribble. Notably, this approach identified a robust network of cell polarity, adhesion and RAS-MAPK signalling proteins that are required for suppression of oncogenic RASV12 transformation mediated by Scribble. In direct support of these findings, a parallel analysis of the transcriptome of cells expressing RASV12 and Scribble, either alone or in combination, revealed that this network of RAS suppression cell polarity proteins are targeted for transcriptional deregulation by RAS via an ERK-FRA1 dependent mechanism. Conversely, enforced Scribble expression was sufficient to restore basal expression of these polarity genes. Together these findings demonstrate for the first time bidirectional feedback loops that serve to integrate cell polarity with RAS-MAPK signalling in the context of oncogenic transformation. Additionally, a novel role was identified for a TPL2-MAPK-JNK2-JUND stress signalling pathway in iv Scribble-mediated suppression of morphological transformation driven by RAS, suggesting that Scribble controls a human cell polarity network that constitutes a novel form of balancing crosstalk between MAPK-ERK and MAPK-JNK signalling to regulate epithelial polarization and suppress the RAS oncogene. Finally, to examine whether regulation of ERK signalling was a common property of all cell polarity regulators, I designed and applied a comprehensive siRNA library corresponding to the known human cell polarity genes and assessed their role in regulation of the sustained phase of ERK signalling using a high-content bio-image analysis screen. This analysis revealed a previously unappreciated degree of complexity and specificity regarding polarized regulation of ERK signalling, and identifies the polarity network as a novel class of highly specific spatiotemporal modulators of the ERK signalling response. Through this combination of systems-level RNAi mediated approaches, I have been able to construct a detailed working map of the cross-regulation and interplay between the RAS-MAPK-ERK signalling pathway and the broad network of genes that regulate epithelial polarity. This map will now serve as a foundation for future work aimed at understanding the functional landscape of human epithelial polarity networks in normal and malignant cells. Such future studies will serve to bridge the gap in our understanding of how polarity networks and oncogenic signalling interact to promote cancer progression.
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ItemGeneration of novel chimeric antigen receptors to enhance the specificity and activity of T cells for the adoptive immunotherapy for cancer( 2014)Adoptive immunotherapy is a promising treatment for cancer, with response rates of up to 70% in metastatic melanoma. To broaden this approach, T cells have been genetically modified to express chimeric antigen receptors (CARs) to endow T cells with anti-tumour activity capable of recognising a range of different cancer types. This approach has shown encouraging results in recent clinical trials for the treatment of haematological malignancies, however it has shown only moderate activity against solid cancers. To date, only a small number of molecules involved in T cell signaling have been incorporated into CARs, resulting in their suboptimal activity. Therefore improvements in CARs are needed in order to realise the full potential of adoptively transferred T cells. We proposed that using multiple or alternate signaling domains could enhance CAR-mediated T cell function. In this thesis, we describe the use of a DNA library of signaling molecules to investigate novel combinations of signaling molecules that could mediate enhanced CAR activity in the Jurkat T cell line and primary human T cells. A novel single-chain variable receptor was discovered comprising DAP10, CD3ζ and CD27 signaling domains that was able to trigger enhanced T cell activity in vitro and in an adoptive transfer mouse model. Clinical trials utilising CAR modified T cells have in some cases resulted in resulted in severe autoimmunity due to T cell recognition of tumour-associated antigens expressed on normal tissues. It is anticipated that as the application and efficacy of adoptive immunotherapy increases, toxicity against normal tissue will become increasingly common. To address this, we proposed that a T cell will respond less against normal tissue if endowed with a tumour-associated antigen-specific activating CAR co-localised with a chimeric inhibitory receptor (CIR) that is capable of turning off the T cell following engagement of antigen on normal tissue. We generated several novel chimeric inhibitory receptors and demonstrated expression of both CAR and CIR in T cells, which were then characterised for function against tumour-associated and normal tissue antigen expressing cell lines. In conclusion, the combination of these novel chimeric receptors may lead to a more efficacious but safer therapy for cancer.