Finch, Andrew

Wilkinson, Simon

Pantazi, Asimina

2018-07-24T11:55:28Z

2018-07-24T11:55:28Z

2018-06-30

Ribosome biogenesis is an essential biological process that is required for cell division and growth. Cancer cells alter their physiology in order to meet their excessive growth demands and therefore maintain abnormal metabolism and homeostasis. Under normal conditions, ribosome biogenesis is tightly regulated to maintain adequate ribosomal content of the cell. However, several oncogenes promote this process and elevated ribosome biogenesis is often found in cancer cells, where it can support the high biosynthetic demand of these cells. Hence, ribosome biogenesis is a process that might provide candidate targets for therapeutic intervention. The main aim of this research was to assess whether inhibition of late stage biogenesis of the 60S ribosomal subunit would result in tumour suppressive responses in normal and cancer cells. We focused upon two GTPases, EFL1 and LSG1, that catalyse the last two cytoplasmic reactions in the maturation of the 60S subunit. We observed that RNAi-based silencing of the GTPases in human lung fibroblasts triggered growth arrest and senescence, which was mediated by the p16 and p53 pathways. Inhibition of these pathways revealed that loss of p53 could bypass the senescence response. However, when cells were plated at low density, knockdown of LSG1 conferred a tumour suppressive response, even in the absence of p53. Knockdown of LSG1 in MCF-10A mammary epithelial cells that lack the p16 locus also induced a robust senescence response and this was also observed in transformed derivatives of MCF-10A cells. Preliminary data obtained in a 3D mammosphere culture model also revealed that inhibition of 60S maturation could elicit an antiproliferative response. Taken together, these data indicate that at least some cancer cells would be responsive to a therapy based upon inhibition of 60S subunit biogenesis. We further characterised the senescence response that was obtained through knockdown of LSG1 by performing gene expression analysis. This revealed a minimal Senescence-Associated Secretory Phenotype (SASP) that was restricted to members of the TGF-β family and lacked the canonical pro-inflammatory cytokines and chemokines that are found in the SASP of cells undergoing oncogene-induced senescence (OIS). Surprisingly, we also observed a dramatic increase in expression of multiple genes in the cholesterol biosynthesis pathway, although inhibition of this pathway indicated that cholesterol biosynthesis was not required for the senescence response. Further insight into the mechanisms of induction of the ribosomal stress senescence response was sought through pilot CRISPR screen and reverse phase protein array (RPPA) analyses. These revealed some interesting leads that will direct future studies.

http://hdl.handle.net/1842/31416

en

The University of Edinburgh

ribosome

senescence

cancer

translation

proliferation

LSG1

Understanding tumour suppressive responses upon inhibition of ribosome maturation

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy