Utilising Yeast as a Model Organism to Deconstruct the Regulation of Tumour Associated Lipogenesis

Abstract

It is important for cells to respond to external signals. Central to these responses are the sensing and signalling pathways that communicate with the nucleus and facilitate necessary changes in gene expression. Of particular importance are the mitogen-activated protein kinase (MAPK) and the mammalian target of rapamycin (mTOR) pathways. Both of these pathways have been shown to be involved in cell growth, proliferation, motility and survival. They are under intensive investigation in connection with cancer with recent evidence suggests their role in mediating lipogenesis. Lipogenesis accompanies a variety of disease states, including the formation of brain tumours. Malignant brain tumours are rapidly growing and often invade surrounding healthy tissue, resulting in poor prognosis for the patient. The ability to limit tumour growth and reduce invasion through a better understanding of tumour associated lipid formation may offer targets for the development of new therapies.
Yeast is frequently used as a paradimic organism for the study of human diseases. In this study a Nile red assay has been developed, optimised and validated to measure levels of both polar and neutral lipids within yeast cells. This method has been utilised in the yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe, to study the role of the MAPK pathways in regulating lipid accumulation. Data in this thesis demonstrates that stress-activated protein kinase pathways (SAPK) play a key role in regulating lipid accumulation upon nitrogen limitation, as cells enter the stationary phase of growth. Evidence from S. cerevisiae proposes that the lipogenic switch occurs in two phases, with the central MAPK (Hog1) activated in both a MAPKK (Pbs2) independent and dependent manner. Analysis of Hog1 phosphorylation during various growth phases, suggests that there are previously uncharacterised sites on Hog1 which are potentially phosphorylated during phase one by the protein kinase Sch9, a target of the Tor1 complex. The second phase results in Hog1 being dually phosphorylated by the canonical pathway, via Pbs2p. It is proposed that Hog1 may have a number of downstream cytoplasmic and nuclear targets, including lipid related enzymes (Dga1) and transcription factors (Msn2/4). Data also suggests that lipid accumulation in S. pombe is also regulated in a similar manner.
The oleaginous yeast Lipomyces starkeyi is able to accumulate high levels of lipid and has similarity to lipid enzymes found in mammalian cells. As such, it was proposed that L. starkeyi may be utilised as a model organism to further characterise the role of MAPK in lipid accumulation. Information from stress response studies and bioinformatics suggests the MAPK pathway in L. starkeyi is highly conserved. However, the application of yeast molecular tools to L. starkeyi was unsuccessful, demonstrating that further work is required to develop its use as a model organism.
Data in this thesis has shown a novel role for the SAPK pathways in regulating lipid accumulation in yeast. It has also demonstrated cross talk between the MAPK and TOR pathways, resulting in an integrated cellular response. The high level of conservation of these pathways across species, suggests that directly targeting these pathways in cancer cells may reduce tumour associated lipogenesis, therefore inhibiting growth of glioma. With current treatments only delivering limited results, this could help extend patient survival.