AbstractBackground: The alteration of Epidermal Growth Factor Receptor (EGFR) plays a major role in the development of Head and Neck cancer. However, the only FDA approved EGFR target for this neoplasm is the drug Cetuximab. Cetuximab can only be used where there is an over-expression of EGFR. Cetuximab works by competing with the ligands Epidermal Growth Factor (EGF) and Transforming Growth Factor alpha (TGFα) to bind the extracellular domain of EGFR. Inhibition of this ligand binding blocks the activation of subsequent signalling pathways. It is known that not all Head and Neck cancers over-expressing EGFR respond to Cetuximab. It has been postulated that some of these tumours have additional genetic lesions. Resistance to Cetuximab has been associated with the genetic lesions to EGFR mutation and Ras mutations. These mutations are more common in Asian than Caucasian patients. More recently the Tyrosine kinase inhibitors (Gefitinib and Erlotinib) have been investigated as drugs to target tumours with EGFR mutation. These inhibitors bind to the cytoplasmic domain of the EGFR and compete with ATP and inhibit phosphorylation of the receptor. Another inhibitor of interest is PD98509 which is a selective ERK1/2 inhibitor and may be used in targeting tumours with a Ras mutation. None of these inhibitors has received FDA approval for Head and Neck cancer treatment.
Aim: The main aims of the project were to investigate the effect of the growth factors EGF and TGFα and the inhibitors; Gefitinib, Erlotinib and PD98059 on cancer cell lines obtained from Asian head and neck cancer patients. The first area of study was to investigate the effect of the growth factors EGF and TGFα on cell proliferation, morphological changes, EMT marker expression, single cell migration and collective cell migration. Once the initial data had been collected a second part was to determine whether the inhibitors; Gefitinib, Erlotinib and PD98059 could block the stimulatory effect of EGF and TGFα in these assays.
Methods: Three tumour cell lines of Asian origin were used in the study and a normal skin keratinocyte as a control. Cell proliferation was measured using an automated cell counting system. Migration was studied using a cell scatter and cell scratch assay. The movement of the cells was documented by using photomicrographs. Epithelial to Mesenchymal Transition (EMT) and protein expression were determined using immunofluorescence and SDS-PAGE and Western blotting.
Results: Cell proliferation data indicated that the growth factors did not stimulate the proliferation of the cells investigated.
Addition of the growth factors for 48 hours induced an EMT like morphological change, individual cell migration in the scatter assay and collective cell migration in the scratch assay. However, the specific markers of EMT (E cadherin and vimentin) as measured by Western blotting and immunofluorescence showed no change in intensity following the addition of the growth factors for 48 hours.
Pre-treatment of the cells for one hour with the tyrosine kinase inhibitors (Gefitinib, Erlotinib) and the MAPK inhibitor (PD98059) inhibited these morphological changes, and individual cell migration. However, only the Tyrosine kinase inhibitors inhibited collective cell migration. PD98059 partially inhibited collective cell migration of the TYS and HaCaT cell lines and fully inhibited cell migration of the HSG and AZA1 cell lines.
Analysis of the MAPK pathway using the expression of phosphorylated MAPK was investigated by Western Blot and Immunofluorescence. Western blot data indicated an increased expression of phosphorylated MAPK202/204 was observed in cells treated with both EGF and TGFα for 24 hours. At the 24hour time point, tyrosine kinase inhibitors (Gefitinib, Erlotinib) fully inhibited the MAPK202/204 phosphorylation. In contrast, in ERK1/2 inhibitor (PD98059) treated dishes, phosphorylated MAPK 202/204 was still expressed which was likely to be the same level of expression as serum free treated dishes.
Conclusion and Possible Clinical Impact: The data indicates that the growth factors EGF and TGFα both stimulated single and collective cell migration. The data lead us an unexpected answer as to whether single cell migration and collective cell migration utilised the same signalling pathways. The data indicated that these are separate pathways; the MAPK pathway appears to be the only pathway responsible for morphological change and single cell migration in cell lines used in this project. The role of MAPK signalling pathway in collective cell migration appears to be cell line dependent and MAPK alone might not be responsible for collective cell migration. The ability of the inhibitors may in the future lead to a more personalised therapy for patients in Asian countries and more clinical trials across a diverse group of patients is needed.
|Date of Award
|Ian Ellis (Supervisor) & Peter Mossey (Supervisor)