Table of Contents:
Cancer is the fourth leading cause of death in Germany with a lifetime prevalence of 45% in men and 38% in women. 90% of the malignancies in the upper aerodigestive tract are squamous cell carcinoma (Head and Neck Squamous Cell Carcinomas, HNSCC). The main risk factors are smoking and drinking alcohol. The epidermal growth factor receptor (Epidermal Growth Factor Receptor, EGFR) shows overexpression in up to 100% of this carcinomas and correlates with the prognosis of the disease. As a result EGFR is the aim of different therapeutic agents such as monoclonal antibodies (e.g. Cetuximab) or EGFR specific kinase inhibitors (e.g. Gefitinib, Erlotinib). To investigate the EGF receptor and his signaling pathways it is therefore very important to understand the biology of HNSCC.
As EGFR activation correlates with the course of illness it was of interest to investigate how sensitiv tumor cells from different HNSCC react on an incubation with the EGFR ligand EGF. To investigtae this well establishes HNSCC cell lines were incubated with rising concentrations of EGF (0; 0,1; 1; 10; 100; 1000 ng/ml) for 12h. Detection of the phosphotyrosin 1173 revealed an increase in EGFR phosphorylation. Interestingly there were dramatic differerences between the different HNSCC cell lines concerning the sensitivity of receptor phosphorylation at ligand incubation. HNSCC cell line UMB-SCC-864, for example, showed maximal recptor phosphorylation at incubation with 0,1 ng EGF/ml whereas UT-SCC-26A showed only weak phosphorylation even at 1000 ng EGF/ml. Transferred to the situation in vivo this findings show a heterogene sensitivity of the EGF signalling pathways. This correlates to heterogen clinical findings concerning the response rates of patients to anti-EGFR therapies.
Cell incubation with EGF leads to internalisation and degradation of the EGF-receptor in co-localisation with AP2. At the same time cells change from a spread out to a spheric morphology.
During a pilot experiment the HNSCC cell line UM-SCC-3 showed a massiv up-regulation of the cell cyclus inhibitor p21WAF1/CIP1 after incubation with 100 ng EGF/ml in a gene expression analysis. Those findings could be confirmed on protein levels on nearly all used HNSCC cell lines using 100ng EGF/ml. To rule out that this apparently paradox effect could be, as assumed by another group, the result of unphysiologically high EGF concentrations (100 ng/ml) p21WAF1/CIP expression was observed via western blot analysis and quantitative real-time PCR using rising concentrations of EGF (0; 0,1; 1; 10; 100; 1000 ng/ml). Both methods showed a direct connection between increasing expression and rising EGF concentration. Protein and RNA concentrations showed tight correlations. At incubation with extremely high EGF concentrations (1000 ng/ml) there was a slight stagnation of EGFR protein and RNA expression. This might be due to a ligand (EGF) induced depletion of the EGF-receptor from the cell surface. Incubation of the HNSCC cells with the EGFR specific kinase inhibitor AG1478 not only leads to a reduction in basal EGFR phosphorylation but also to a reduction in basal p21WAF1/CIP1expression. Those findings support the hypothesis that p21WAF1/CIP1 expression correlates with the activationstatus of EGFR.
In summary the manuscript in hand displays data showing a high variabilty in the receptor activation in HNSCC. It therefore seems probable that anti EGF strategies (Cetuximab, Gefitinib, Erlotinib) have very different response rates as can also be observed in clinical practice. Future studies should therefore not only observe the EGFR recptor expression but also EGFR activation levels as a possible prognostic marker. The interesting but seemingly paradox observation of a direct correlation of EGFR activation and p21WAF1/CIP1 expression needs to be further investigated concerning its importance in cell cycle and radio and chemo sensitivity of HNSCC cells.