Quantification of spatiotemporal patterns of Ras isoform expression during development

Ras proteins are important signalling hubs frequently dysregulated in cancer and in a group of developmental disorders called Rasopathies. Three Ras genes encode four proteins that differentially contribute to these phenotypes. Using quantitative real-time PCR (qRT-PCR) we have measured the gene expression profiles of each of the Ras isoforms in a panel of mouse tissues derived from a full developmental time course spanning embryogenesis through to adulthood. In most tissues and developmental stages we observe a relative contribution of KRas4B > > NRas ≥ KRas4A > HRas to total Ras expression with KRas4B typically representing 60-99% of all Ras transcripts. KRas4A is the most dynamically regulated Ras isoform with significant up-regulation of expression observed pre-term in stomach, intestine, kidney and heart. The expression patterns assist interpretation of the essential role of KRas in development and the preponderance of KRas mutations in cancer

Isoform-specific Ras expression and signalling.

Ras proteins are GTPases that are molecular central hubs for propagation of intracellular signals that are involved in diverse processes, including cell differentiation and proliferation. The four main Ras isoforms, HRas, NRas and the two KRas splice variants, KRasA and KRasB, are highly homologous and conserved proto-oncogenes that constitute a paradigm of cellular transformation. While all RAS genes are commonly mutated in human cancers, different isoforms couple to distinct tumours and the mutations in KRAS constitute the majority (~86%) of all RAS mutations. Intriguingly, KRasB is the only Ras isoform essential for normal embryonic growth in the mouse. Such discrepancy of Ras isoform contribution to different cancers and the uniquely essential role of KRasB in normal development may stem from the distinct spatiotemporal expression of Ras isoforms and their differential coupling to downstream effectors. However, to date there has been no comprehensive quantitative comparison of Ras isoform expression across various tissues throughout development. Moreover, there are no studies that compared Ras isoform-specific signalling in an endogenous context. Therefore, this thesis aims to provide the first complete map of Ras isoform expression during development and the first comparison of endogenous Ras isoformspecific signalling. In the first part of this work, quantitative real-time RT-PCR was used to measure Ras isoform transcript levels in mouse embryonic stem cells (ESCs) and in a panel of embryonic, postnatal and adult mouse tissues. KRasB was found to be the most highly expressed isoform, whereas KRasA was shown to be the most dynamically regulated. Transcript copy number does not necessarily correlate with protein copy number; therefore, protein standard absolute quantitation mass spectrometry was used to accurately measure tissue Ras protein levels. In contrast to the qRT-PCR data where KRasB was 5-10-fold higher expressed than any of the other isoforms, protein abundance levels were found to be similar for the Ras isoforms. The mechanistic basis for this and the implications for models of isoform-specific Ras association with specific cancers are discussed. In the second part of this thesis, isogenic SW48 human colorectal cancer cell lines identical except for the presence of an activating G12V mutation in each of the three RAS gene loci were utilised to study endogenous Ras signalling. The results revealed isoform-specific coupling to canonical Ras effector pathways for example, HRas was the most potent activator of downstream MAPK and PI3K pathways. These data represent the basis for planned network biology studies to model Ras isoform-specific signalling. Together, this study provides the first most complete approaches for studying baseline Ras isoform expression and signalling in an endogenous context. The results guide our understanding of Ras isoform-specific network biology, coupling to distinct human cancers and involvement in normal development

Interaction of galectin-3 with MUC1 on cell surface promotes EGFR dimerization and activation in human epithelial cancer cells

Epidermal growth factor receptor (EGFR) is an important regulator of epithelial cell growth and survival in normal and cancerous tissues and is a principal therapeutic target for cancer treatment. EGFR is associated in epithelial cells with the heavily glycosylated transmembrane mucin protein MUC1, a natural ligand of galectin-3 that is overexpressed in cancer. This study reveals that the expression of cell surface MUC1 is a critical enhancer of EGF-induced EGFR activation in human breast and colon cancer cells. Both the MUC1 extracellular and intracellular domains are involved in EGFR activation but the predominant influence comes from its extracellular domain. Binding of galectin-3 to the MUC1 extracellular domain induces MUC1 cell surface polarization and increases MUC1–EGFR association. This leads to a rapid increase of EGFR homo-/hetero-dimerization and subsequently increased, and also prolonged, EGFR activation and signalling. This effect requires both the galectin-3 C-terminal carbohydrate recognition domain and its N-terminal ligand multi-merization domain. Thus, interaction of galectin-3 with MUC1 on cell surface promotes EGFR dimerization and activation in epithelial cancer cells. As MUC1 and galectin-3 are both commonly overexpressed in most types of epithelial cancers, their interaction and impact on EGFR activation likely makes important contribution to EGFR-associated tumorigenesis and cancer progression and may also influence the effectiveness of EGFR-targeted cancer therapy

Chlamydophila psittaci-negative ocular adnexal marginal zone lymphomas express self polyreactive B-cell receptors

Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease