Gene targeting in lung cancer

Abstract

Lung cancer is one of the leading causes of cancer death in the world, whilst being the most common malignancy in men and third most common cancer in women. One of the main deregulated pathways within lung cancer is the mTOR pathway. The downstream effects of this deregulation causes high levels of free eIF4E within the cell, promoting oncogenic networks such as increased proliferation, metastasis and cell survival. This project aimed to investigate the effects of eIF4E inhibition in NSCLC, both by treating commercial cell lines with small-molecule eIF4E inhibitors and also investigating the effects of knockdown by siRNA of eIF4E. Using three different cell lines, each with unique characteristics depending on their subtype of NSCLC were used throughout the project. The two small-molecule inhibitors were Briciclib and 4E1RCat, where Briciclib showed a large and significant decrease in cell viability at low concentrations throughout the cell lines, as opposed to 4E1RCat which did not show significant decreases in cell viability when treating with low dosages. ELISA assays were then performed on cells treated with Briciclib by investigating the effects of the drug on Cyclin D1, which is found downstream of eIF4E. Results showed a decrease in Cyclin D1 expression. Finally, transfections were used to knockdown eIF4E through an siRNA. Conditions were optimised prior to knockdown of eIF4E using siGLO and flowcytometry. Knockdown was then performed and cell viability assays were used to observe any initial effects on the cell lines but it was seen that there was little to no decrease in cell viability upon knockdown. qPCR was performed and showed large decreases in eIF4E expression. These results were seen to be promising and worth investigating further in the future, such as investigation of the effects of Briciclib in primary cells to investigate the cytotoxicity of the drug further, and also investigating the effects of knockdown of eIF4E along with inhibition through small-molecules to increase the efficacy of the two. Finally, these results confirm that there is a decrease in cap-dependent translation of oncogenes within the cancer cell line, which in turn decreases cell viability and increases cell death.