Friedmann, Benjamin Jacob; (2006) Modulation of chemotherapy by inhibition of the epidermal growth factor receptor. Doctoral thesis , UCL (University College London).

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Abstract

Purpose: The epidermal growth factor receptor (EGFR) is commonly expressed in human tumours and provides an important target for therapy. Several classes of agents including small molecule inhibitors and antibodies are currently under clinical development. These agents have shown interaction with chemotherapeutic agents in vitro and in vivo. However, the mechanisms of these interactions are not clearly understood. The purpose of this study was to investigate mechanisms for this modulation. Experimental Design: The synergistic effects of the EGFR inhibitor gefitinib (Iressa, ZD 1839) in combination with a variety of chemotherapeutic agents was determined in several cancer cell lines and analysed pharmacologically. Using the alkaline single-cell gel electrophoresis (comet) assay, the kinetics of DNA damage and repair following treatment with the chemotherapeutic drugs combined with gefitinib were investigated. The modulation of DNA-PK activity by gefitinib was quantitated using a variety of techniques including immunoprecipitations, immunoblotting, cellular fraction extractions and immunohistochemistry. The effects of inhibiting the DNA repair protein DNA-PK, with LY294002, wortmannin and siRNA to its catalytic subunit (DNA-PKCs) were investigated in the same cancer cell lines and compared with the effects of gefitinib. Results: Synergistic effects of EGFR inhibitors and chemotherapy were found with a variety of cell lines. The synergy was found with etoposide, doxorubicin and cisplatin but interestingly not with melphalan. Experiments on DNA repair using the comet assay demonstrated delay in repair of DNA strand breaks and inter-strand cross links (ICLs) by gefitinib following treatment with etoposide and cisplatin respectively. The mechanism of this interaction was investigated and found that interference with the DNA-PK pathway mimicked the effects of EGFR inhibition. Additionally, gefitinib treatment induced a physical interaction between EGFR and DNA-PK and a cellular re distribution of DNA-PK was associated with a fall in DNA-PK activity. Conclusions: These results highlight the important effects EGFR inhibitors have on DNA repair and its associated machinery when added in combination with certain chemotherapeutic agents. These results will form the design of further clinical schedules combining these classes of agents and point the way to further studies to investigate the molecular mechanisms of these interactions.

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