An investigation into the role of the initiator methionine transfer RNA in cell migration and tumour growth

Clarke, Cassie J. (2015) An investigation into the role of the initiator methionine transfer RNA in cell migration and tumour growth. PhD thesis, University of Glasgow.

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Abstract

Control of cellular tRNA repertoires can drive specific programmes of translation to favour the maintenance of proliferative or differentiated phenotypes. tRNAiMet is the initiator methionine tRNA, responsible for recognising the start codon and initiating translation. We have investigated how increased expression of tRNAiMet can influence cell behaviour, using both immortalised cell lines in vitro and mouse models in vivo. Levels of tRNAiMet are increased in carcinoma-associated fibroblasts compared to normal fibroblasts. To understand the cellular effects of tRNAiMet overexpression in more detail we generated immortalised mouse embryonic fibroblasts (iMEFs) that overexpressed tRNAiMet (iMEF.tRNAiMet) or an empty vector as control (iMEF.Vector). Full characterisation of iMEF.Vector and iMEF.tRNAiMet cell lines showed that overexpression of tRNAiMet did not affect cell size, energy metabolism, cell spreading, rate of cellular protein synthesis or proliferation. Increased expression of tRNAiMet did, however, have marked effects on cell migration; with iMEF.tRNAiMet cells migrating approximately 1.5 fold faster than iMEF.Vector controls when assessing both directional and random migration. This tRNAiMet-driven increase in cell speed was dependent on the levels of phosphorylated eIF2α, indicating that fibroblast migration might be influenced by tRNAiMet in the ternary complex. Furthermore, the ability of tRNAiMet to increase cell migration depended on the ability of integrin α5β1 to bind its extracellular ligand fibronectin. However, despite the robust and reproducible role of both phospho-eIF2α and integrin α5β1 in this process, the way in which these are mechanistically linked to tRNAiMet levels is yet to be determined. To investigate whether increased stromal tRNAiMet expression may contribute to tumour progression, we utilised a mouse that expressed additional copies of the tRNAiMet gene (2+tRNAiMet mouse), and performed syngeneic allografts into these animals. Subcutaneous allograft tumours of a number of different cancer cell lines became more vascularised and grew significantly more rapidly in 2+tRNAiMet mice by comparison with tumours grown in littermate control animals. The extracellular matrix (ECM) that was deposited by fibroblasts from 2+tRNAiMet mice was found to support enhanced endothelial cell and fibroblast migration. We used SILAC mass spectrometry to compare the secretome of iMEF.Vector and iMEF.tRNAiMet cell lines and found that overexpression of tRNAiMet significantly increased synthesis and secretion of certain types of collagen, in particular collagen II. Moreover, knockdown of collagen II using siRNA and CRISPR approaches opposed the ability of tRNAiMet overexpressing fibroblasts to deposit a pro-migratory ECM. We used the prolyl hydroxylase inhibitor, ethyl-3,4-dihydroxybenzoate (DHB), to determine whether collagen synthesis contributed to the ability of tRNAiMet to drive a pro-tumorigenic stroma in vivo. Administration of DHB had no effect on the growth of syngeneic allografts in wild-type mice, but opposed the ability of 2+tRNAiMet animals to support increased angiogenesis and tumour growth. Collectively these data indicate that increased expression of tRNAiMet contributes to tumour progression by enhancing the ability of stromal fibroblasts to synthesise and secrete a collagen II-rich ECM that supports endothelial cell migration and angiogenesis. Taken together these data provide evidence that the tRNAome, and in particular cellular levels of tRNAiMet, influence both the migration of fibroblasts and the composition of their secretome in a way that promotes the generation of a microenvironment supportive of endothelial cell migration, angiogenesis and tumour growth.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Initiator methionine tRNA (tRNAiMet), extracellular matrix, tumour angiogenesis, secretome, tRNA repertoire, cell migration, tumour stroma
Subjects: Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences > Beatson Institute of Cancer Research
Funder's Name: UNSPECIFIED
Supervisor's Name: Norman, Professor Jim
Date of Award: 2015
Embargo Date: 29 November 2017
Depositing User: Cassie Jemma Clarke
Unique ID: glathesis:2015-6913
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 04 Dec 2015 14:10
Last Modified: 11 Dec 2015 13:37
URI: http://theses.gla.ac.uk/id/eprint/6913

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