title: Cellular responses to oncogenic Ras signalling
Creators: name: Mageean, Craig
Publisher: name: University of Liverpool Repository
Year: 2015

description: The three human Ras proto-oncogenes encode for four highly homologous protein isoforms, H-, K(A)-, K(B)- and N-Ras, that function as molecular switches and timers to transduce signals from activated cell surface receptors to modulate signalling pathways responsible for cell proliferation, growth, survival and apoptosis. Oncogenic mutations of Ras occur in ~16% of all human cancer cases and most often affect codons 12, 13 and 61, each resulting in constitutive activation of the protein. Currently, it is uncertain how many Ras molecules are present per cell and which isoform is most abundant, essential information for the emerging field of systems biology, which utilises mathematical modelling to understand the behaviour of complex signalling pathways in a holistic approach. In the first part of this thesis, a selected reaction monitoring (SRM)-based Ras quantification technique is established for the measurement of each major isoform, along with the generation of isotope-labelled Ras protein standards that support quantification using the protein standard absolute quantification (PSAQ) strategy. Combining targeted SRM-based proteomics with the PSAQ strategy enabled the most accurate measurement of cellular Ras isoform abundance to date, as detailed in the second part of this thesis. The target cell lines in the present thesis were a panel of isogenic SW48 colorectal cells, which express a variety of heterozygous Ras mutations following the exchange of a wild-type allele for a mutant Ras sequence through targeted homologous recombination. Over 250,000 Ras molecules per wild-type SW48 cell were quantified, with K(B)-Ras the major Ras isoform. ~114 Ras molecules are predicted to be bound per μm2 of plasma membrane, with the cellular molarity of Ras at 253 mM. Intriguingly, the presence of certain K-Ras mutations induced significant changes in cellular Ras abundance, which may be attributable to the specific transforming potential of each mutant protein. The presented data also suggests that, in addition to their structural and biochemical variations, Ras mutant proteins may exert their different biological effects through differential protein expression. Many cancer types demonstrate a preference for a single Ras isoform to be mutated and present a codon-specific mutation signature. In colorectal cancer, K-Ras is the most frequently mutated isoform, with over 75% of mutations accounted for by G12D, G12V and G13D in colon tumours with a K-Ras mutation. Clinical data suggests that patients harbouring codon 12 or 13 K-Ras mutations have different overall survival rates and responsiveness to treatment. Isogenic SW48 cells harbouring the aforementioned K-Ras mutations were subject to mass spectrometry-based proteome and phosphoproteome quantitative analysis, to investigate the effects of different amino acid substitution (G12D vs. G12V) or codon mutation (G12D vs. G13D) on oncogenic Ras signalling. The presented data in the third part of this thesis provide the first quantitative proteomic and phosphoproteomic profile of signatures associated with specific K-Ras mutant proteins expressed at endogenous levels. Each K-Ras mutation induced a distinct signalling output, with the most variability observed between codon 12 mutants and G13D. This indicates that the position of a Ras point mutation may have more impact on signalling than an amino acid substitution. Several proteins relevant to colorectal carcinogenesis were found to specifically up-regulated by codon 12 or 13 mutations, with one notable example being the recently described colon cancer stem cell marker double cortin-like kinase 1, which was sensitive only to codon 12 K-Ras mutations. Together, the data presented in this thesis provides a novel insight into the behaviour of a range of Ras mutations in a colorectal cancer setting.