Abstract
The expression of epidermal growth factor receptor (EGFR/ERBB1/HER1) is implicated in the progress of numerous cancers, a feature that has been exploited in the development of EGFR antibodies and EGFR tyrosine kinase inhibitors as anti-cancer drugs. However, EGFR also has important normal cellular functions, leading to serious side effects when EGFR is inhibited. One damaging characteristic of many oncogenes is the ability to be expressed in the hypoxic conditions associated with the tumour interior. It has previously been demonstrated that expression of EGFR is maintained in hypoxic conditions via an unknown mechanism of translational control, despite global translation rates generally being attenuated under hypoxic conditions. In this report, we demonstrate that the human EGFR 5′ untranslated region (UTR) sequence can initiate the expression of a downstream open reading frame via an internal ribosome entry site (IRES). We show that this effect is not due to either cryptic promoter activity or splicing events. We have investigated the requirement of the EGFR IRES for eukaryotic initiation factor 4A (eIF4A), which is an RNA helicase responsible for processing RNA secondary structure as part of translation initiation. Treatment with hippuristanol (a potent inhibitor of eIF4A) caused a decrease in EGFR 5′ UTR-driven reporter activity and also a reduction in EGFR protein level. Importantly, we show that expression of a reporter gene under the control of the EGFR IRES is maintained under hypoxic conditions despite a fall in global translation rates.
Highlights
Epidermal growth factor receptor (EGFR, ErbB-1 or HER1) is an important drug target and prognostic indicator in many cancers
While a proportion of this overexpression may be attributable to mutations that cause the transcriptional upregulation of the EGFR gene,[15,16,17,18] it has been shown that that EGFR protein levels are upregulated in response to both hypoxia and the activation of hypoxia-inducible factor 2α without observing either mutational events or changes in EGFR mRNA levels.[19]
Since internal ribosome entry site (IRES)-mediated translation initiation does not involve the binding of the 5′ cap, it is favoured under certain conditions, like hypoxia, that inhibit eukaryotic initiation factor 4E function.[26,27,28]
Summary
Epidermal growth factor receptor (EGFR, ErbB-1 or HER1) is an important drug target and prognostic indicator in many cancers. Since internal ribosome entry site (IRES)-mediated translation initiation does not involve the binding of the 5′ cap, it is favoured under certain conditions, like hypoxia, that inhibit eukaryotic initiation factor 4E (eIF4E) function.[26,27,28] Despite this reduced requirement for eIF4E, it has been demonstrated that the IRESs belonging to the human genes c-myc, N-myc and BiP have a strong requirement for eIF4A function for their expression.[29,30,31] It has been suggested that this requirement indicates that the structure of these IRESs needs ‘remodelling’ by eIF4A before they are able to function, similar to the processing required by the encephalomyocarditis virus IRES.[30,32,33,34]. To determine whether cryptic splicing was occuring (which could lead to functional firefly luciferase transcripts in the absence of IRES activity) Northern analysis was
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