Targeting the Hypoxic Expression of Epidermal Growth Factor Receptor

Abstract

Investigating translation regulation of epidermal growth factor receptor (EGFR) is a growing concern in the context of advanced breast cancer. The 5’ untranslated region (UTR) of EGFR mRNA contains an internal ribosome entry site (IRES) that permits hypoxic expression. This allows targeting of the expression of EGFR in breast cancer cells under hypoxia and other stress conditions. Here we describe the role of the EGFR IRES in mediating protein expression under stress conditions commonly associated with malignant growth including DNA damage. The requirement for DDX3, a DEAD-box RNA helicase, was also investigated in terms of regulating the mRNA translation of EGFR via its 5’ UTR. In addition, this study seeks to obtain data with regards to the feasibility of targeting the hypoxic expression of EGFR using the antisense technology and polymeric delivery systems. The response of the EGFR IRES to different conditions of cell stress and DDX3 was examined utilising bicistronic plasmids in breast cancer cells. In addition, antisense oligonucleotides (ASOs) binding different regions of the EGFR 5’ UTR have been used to target the expression of EGFR in breast cancer cell lines. To deliver these ASOs to hypoxic cancer cells, linear cationic polymers based on poly(β‐amino ester) (PBAE) chemistry were synthesised incorporating the hypoxia- sensitive azobenzene bond. Translation driven by the EGFR IRES responds to specific conditions of cell stress, notably those that induce genotoxic stress, suggesting an important role of the EGFR IRES in mediating translation initiation as a response to moderate DNA damage. The second major finding was that DDX3 stimulates cap-independent translation initiation irrespective to the 5’ UTR sequence. In addition, targeting the EGFR IRES using modified ASOs was found to be ineffective in preventing the internal landing of ribosomes, yet targeting the EGFR mRNA for degradation was found to significantly reduce the expression of EGFR and viability of breast cancer cells. Finally, azobenzene-containing PBAEs were synthesised and characterised showing promising results in terms of their ASO complexation stability and responsiveness to low oxygen tensions. Once established, these PBAEs can be used to deliver the anti-EGFR ASOs preferentially to hypoxic cells in breast-cancer tumours.