Title: Molecular mechanisms regulating traffic of EGF receptor and their role in modulating responses to cancer therapeutics
Funder: Medical Research Council

Abstract: EGF receptor (EGFR) overexpression is associated with tumorigenesis and these receptors are important targets for cancer therapies. Although anti-EGFR therapies have improved treatment of head and neck, lung and colorectal cancers, their activity as monotherapies is low. However EGFR inhibitors can potentiate the activity of chemotherapeutic agents and irradiation. DNA damage following irradiation and chemotherapy activates EGFR independently of ligand and has been reported to promote nuclear translocation of EGFR and DNA repair. We found that cells expressing EGFR with defective nuclear translocation, including mutants found clinically, are unable to efficiently repair chemotherapy and irradiation-induced DNA damage. Endocytosis and subsequent intracellular traffic of EGFR are important regulators of EGFR signalling following ligand stimulation but the trafficking of EGFR activated following DNA damage is poorly understood. Despite its potential importance nuclear translocation of EGFR remains controversial and its molecular regulation is also poorly understood. Ligand stimulation and anti-receptor antibodies, as well as irradiation and chemotherapies, have been reported to promote nuclear EGFR translocation, but the relationship between trafficking pathways following these different stimuli is unclear. We will determine the time course of nuclear traffic of wild type and mutant EGFR (found in tumors and lacking the putative nuclear localisation sequence) in response to ligand, irradiation and chemotherapies in the presence/absence of EGFR inhibitors and correlate this traffic with efficiency of DNA repair. We will then comprehensively describe the pathways followed by wild type and mutant EGFR following activation by ligand and by irradiation and chemotherapy under conditions where nuclear transport is promoted/inhibited. Finally we will identify molecular mechanisms regulating nuclear transport of EGFR and determine the role of those mechanisms in modulating DNA repair. Fractional immunoblotting, immunofluorescence, immuno-electron microscopy and live cell-imaging of fluorescently tagged EGFR and EGFR mutants will, together, provide unequivocal identification of EGFR localisation. A Rab siRNA library and individual siRNAs and small molecule inhibitors targeting candidate proteins implicated in nuclear import will be used to identify molecular regulators of this process. Effects on DNA repair will be measured using comet and H2AX assays. These studies will directly compare trafficking pathways followed by EGFR activated ligand-dependently and independently and will identify fundamental mechanisms regulating transport of a transmembrane protein into the nucleus. They will determine how nuclear transport of EGFR modulates response to cancer therapy, will provide a mechanistic basis to predict likely responses to therapy and design improved combinatorial therapies and may identify novel therapeutic targets.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS