Global proteome response of human cancer cell lines to low dose eIF4E/eIF4G inhibition

Cachexia is a debilitating muscle wasting disease and co-morbidity strongly associated with chronic inflammatory conditions such as cancer, chronic heart failure, chronic obstructive pulmonary disease and sepsis. Cachexia has a strong negative impact on quality of life and research suggests that 20% of cancer patients will die of cachexia. Translation initiation is the most highly regulated step of protein synthesis and the eukaryotic initiation factor 4F (eIF4F) translation initiation complex is the gatekeeper of this process; the eIF4F complex is composed of eIFG, a scaffolding protein, eIF4E, an mRNA cap-recognition protein and eIF4A, an RNA helicase. Inhibition of eIF4A by pateamine A has been shown to rescue muscle wasting in vitro and in vivo, this result has been reproduced with other eIF4A inhibitors. Pateamine A is a sponge-derived natural product with nanomolar toxicity to cancer cells. Surprisingly, at doses well below its anti-neoplastic activity it exerts distinct effects on cachexia. The research in this thesis follows on from previous work in our laboratory with pateamine A in human cell lines. Work on the effects of pateamine A on the proteome suggests that not all the proteins changing in expression are explainable by stressing the translation initiation complex. A model by which motifs in the 5’ UTRs of transcripts are a recognised and removed from the system in a selective manner could help explain these effects. We aimed to target eIF4E, another component of the eIF4F system, with two compounds to see if a comparable dose of eIF4E inhibitors could elicit a pateamine-like response. DMSO, a solvent used extensively in this thesis, had unexpected effects on translation. We conclude that 4E1RCat, a compound developed as a selective inhibitor of eIF4E, is not likely to be useable in further work, due to its window of activity coinciding with an unacceptable concentration of DMSO. Ribavirin, our second compound, showed a proteomic response consistent with its classification as an eIF4E translation initiation inhibitor. The proteome response seen with our eIF4E inhibitors is consistent with disruption of translation initiation. However, the data for 4E1RCat was deemed untrustworthy in the wake of revelations that DMSO, the vehicle in which it is dissolved, exerts an almost identical response. From the results obtained, it was not possible to confidently test whether protein downregulation occurred in response to a 5’UTR sequence motif, as seen for inhibitors of eIF4A. Coupled with the uncertainty associated with the 4E1Rcat results, there were relatively few downregulated proteins from the treatments, and many of these could be explained by the direct biological response to the function of the compound in the treatment. All in all, we have obtained new insights into the effects of DMSO on the proteome which will aid further experimentation. This thesis has laid the groundwork for further investigation of the effects of eIF4F inhibition in the context of better understanding the remediation of cachexia through the eIF4F system.