Vaughan, SOK; (2015) The relationship between stress-induced endocytosis of epidermal growth factor receptor and cell survival. Doctoral thesis , UCL (University College London).

Abstract

The epidermal growth factor receptor (EGFR) is best known for its role in triggering a variety of intracellular signalling pathways that promote cell growth, proliferation, differentiation and migration following the binding of its ligand (EGF). As a result of this, over-activation of EGFR can contribute to carcinogenesis. In parallel with receptor activation, ligand binding initiates receptor internalisation, which provides an important mechanism for negatively regulating the duration and potency of signalling by kick-starting EGFR traffic to the lysosome where it is degraded. However, a small proportion of EGFR can be recycled back to the plasma membrane, prolonging signalling. Another controversial fate has been suggested for the EGFR: traffic to the nucleus where it is believed to promote cell cycle progression, proliferation and DNA repair. In addition to ligand-dependent activation and internalisation of EGFR, these processes can also occur in a ligand-independent manner, following various stresses, including chemotherapy, radiotherapy and ultraviolet light C (UVC). The fate of EGFR following ligand-independent stimulation has not been fully characterised, but in some instances it has been reported to traffic to the nucleus. This would have therapeutic implications, as DNA damage following chemotherapy or radiotherapy could be repaired, resulting in the development of resistance to therapy. In this thesis I aimed to establish tools that inhibited stress-induced EGFR endocytosis in order to test the hypothesis that the fate of stress-induced EGFR regulates EGFR signalling, DNA repair and cell survival. I focused on UVC-induced internalisation, as this occurs rapidly and synchronously and shares a mechanism of internalisation with the chemotherapeutic drug cisplatin. I have shown that following UVC irradiation or cisplatin treatment of HeLa cells, EGFR is internalised and retained in a peri-nuclear compartment without being degraded or translocated to the nucleus. This internalisation depends on the activity of both p38 and Src but not the EGFR tyrosine kinase. Electron microscopy revealed EGFR to be sequestered in multivesicular endosomes/bodies (MVBs). Although these MVBs were morphologically indistinguishable from those that traffic ligand-stimulated EGFR to the lysosome, stress exposed EGFR showed little degradation despite apparent near normal lysosomal function. Stress-induced EGFR endocytosis was not directly linked to DNA damage as ionizing radiation and UVA treatment, which induced extensive DNA damage but did not activate p38, did not induce detectable EGFR internalisation in HeLa cells. EGFR internalisation also correlated with subsequent cell death, as only doses of UVC that induced cell death also induced EGFR endocytosis. Stress-induced EGFR internalisation was abrogated upon depletion of the clathrin adaptor complex AP-2, or mutation of the two AP-2 interaction motifs in the cytoplasmic domain of the EGFR, indicating that this endocytosis occurs via clathrin-coated pits. Inhibition of stress-induced EGFR endocytosis demonstrated that endocytosis of EGFR was necessary to maintain EGFR tyrosine kinase activity, downstream signalling and delay entry into apoptosis. In conclusion we have uncovered a potential role for EGFR in the development of therapeutic resistance. By understanding the molecular mechanisms affecting the endocytic fate of EGFR following stress exposure, it may be possible to design strategies that divert patients from such resistance in the future.

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