eIF4A2 is a regulator of hypoxic translation and colorectal tumour cell survivalTools Bolland, Hannah (2020) eIF4A2 is a regulator of hypoxic translation and colorectal tumour cell survival. PhD thesis, University of Nottingham.
AbstractColorectal cancer is the third most common cancer, leading to 700,000 deaths a year worldwide. Regions of hypoxia (<1% O2) are present in around 50% of colorectal tumours. Hypoxia is associated with resistance to chemotherapy, radiotherapy and worse patient outcome. The process of protein synthesis, in particular, translation initiation, is a tightly regulated process orchestrated by the interaction between the mRNA strand, ribosome, RNA binding proteins and eukaryotic initiation factors. Hypoxia reprograms protein output by altering mRNA translation efficiency and components of the protein synthesis machinery to aid cell survival. eIF4A2 is an ATP dependent RNA helicase required to unwind complex secondary structures present in the 5’UTR of transcripts to allow for translation to occur. eIF4A2 is differentially expressed in a variety of cancers and has previously been shown to directly interact with components of miRNA repression machinery. It was therefore hypothesised that eIF4A2 plays a role in normoxic and hypoxic colorectal cancer cell survival through its regulation of translation of proteins required for hypoxic survival. This thesis aimed to investigate the hypoxic regulation and expression of eIF4A2 in colorectal cancer cells in addition to the effect of eIF4A2 knockdown on the hypoxic survival of colorectal cancer cells. A final aim of this thesis is to identify the differentially expressed transcripts bound to eIF4A2 under normoxic and hypoxic conditions in colorectal cancer cells. A variety of cell-based assays were used to identify that eIF4A2 mRNA expression is increased under hypoxic conditions across a panel of colorectal cancer cell lines. Furthermore, eIF4A2 was found to be under the transcriptional control of HIF2α at both the RNA and protein level in two colorectal cancer cell lines. eIF4A2 was found to be required for hypoxic colorectal cancer cell survival in both 2D and 3D assays, this effect was found to be tumour specific as no negative effect on cell survival was observed in a normal colon epithelial cell line following eIF4A2 knockdown. A global RIP-SEQ approach was used to uncover novel transcripts bound to eIF4A2 and differentially expressed between normoxia and hypoxia. Such transcripts up regulated in hypoxia in comparison to normoxia when bound to eIF4A2 were found to be enriched for pathways including metabolism, endocytosis, carbon metabolism, focal adhesion and lysosome along with many other pathways in line with the major changes in hypoxia. 5’UTR analysis revealed that hypoxic eIF4A2 bound transcripts possessed longer and more thermodynamically stable 5’UTRs. This suggests a higher dependence on eIF4A2 to be efficiently unwound. The work presented in this thesis for the first time demonstrates that colorectal cancer cells require eIF4A2 for their survival in both normoxia and hypoxia. Furthermore, the work presented in this thesis uncovers novel hypoxic eIF4A2 bound transcripts and novel pathways potentially regulated by eIF4A2 in hypoxic conditions. These results lead us to the hypothesis that eIF4A2 is a master regulator of the molecular adaption to hypoxia, enabling cell proliferation and survival in hypoxia by regulating the translation of proteins in amino acids synthesis and endocytic pathways.
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