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Modulation of DNA Strand Break Induction and Repair by Tyrosine Kinase Inhibitors Targeted Against EGFR and HER2

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Modulation of DNA Strand Break Induction and Repair by Tyrosine Kinase Inhibitors Targeted Against EGFR and HER2 Modulation of DNA strand break induction and repair by tyrosine kinase inhibitors targeted against EGFR and HER2 Jaishree Bhosle A thesis submitted to the University College London for the degree of Doctor of Philosophy June 2011 CRUK Drug-DNA Interaction Research Group UCL Cancer Institute Paul O’Gorman Building 72 Huntley Street, London, WC1E 6DD, UK Signed declaration I, Jaishree Bhosle confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Purpose The human epidermal growth factor receptors EGFR (erbB1) and HER2 (erbB2/neu) are involved in mediating resistance to chemotherapy and ionising radiation (IR). In vitro studies demonstrate that small molecule tyrosine kinase inhibitors (TKIs) which target these receptors can increase the effectiveness of DNA damaging agents. However, these combinations have failed to produce the clinical results anticipated and one potential explanation is that the inhibition of EGFR and HER2 cell signalling pathways by TKIs is short lived, with cells able to switch to alternative mechanisms of signalling through HER3. The purpose of this study was to examine whether the duration of exposure to TKIs modulates the induction and repair of DNA damage produced by chemotherapy or IR and describes attempts to elucidate the role of HER2 in mediating resistance to chemotherapy. Experimental design Two HER targeting TKIs, lapatinib and gefitinib were investigated. The effect of lapatinib in combination with cisplatin and doxorubicin on the inhibition of cell proliferation and the role of schedule were examined in drug combination assays. The influence of the duration of exposure to TKIs on the induction and repair of DNA lesions induced by cisplatin, IR, doxorubicin, etoposide and m-AMSA were investigated using the alkaline and neutral Comet assays and measurement of γH2AX and RAD51 foci. DNA expression arrays were used to identify the potential mechanisms through which HER2 produces resistance to cisplatin in cells transfected with HER2. Results Lapatinib is able to synergistically inhibit cell proliferation in combination with cisplatin or doxorubicin in a schedule dependent manner. Duration of exposure to TKIs has no effect on the induction of DNA lesions by cisplatin or IR, but significantly reduces the production of DNA double strand breaks by doxorubicin, etoposide and m-AMSA in part through the down-regulation of the expression of topoisomerase IIα (Topo IIα), increasing resistance to these drugs. Conclusions These results indicate the scheduling of small molecule TKIs targeted against EGFR and HER2 is important and continuous exposure to these drugs induces resistance to doxorubicin, etoposide and m-AMSA, through reduced expression of their target, Topo IIα. The importance of schedule should be considered when combining TKIs with chemotherapy in clinical practice. 3 ACKNOWLEDGEMENTS This work was carried out under the supervision of Professor Daniel Hochhauser, Professor John A Hartley and Dr Andreas Makris and funded by the Breast Cancer Research Trust. I would like to thank Daniel for his support, guidance and friendship during the last four years, which continues. John for his advice and helping me to stick to the science and Andreas for his enthusiasm, encouragement and coffee. I would also like to thank all three for their advice over the last four years and in the preparation of this manuscript. I would specifically like to thank Dr J Wu for undertaking the staining, visualisation and counting of the RAD51 and γH2AX foci, Dr P Dhami for undertaking the chromatin immunoprecipitation and Mr J Bingham for performing the RT-PCR. I am grateful to all my colleagues in the laboratory for putting up with me, as well as teaching and helping me; specifically Giammy, Raisa, Samir, Kostas and Valeria. Most of all I am grateful for those who believe in me, especially my parents, brother Amit, sister Rajeshree and my best friends, Bianca and Anna, for providing coffee, cakes and gin PRN. 4 COMMUNICATIONS PRESENTATIONS July 2009 Interaction of HER Inhibition and Chemotherapy in Breast Cancer. UCL Cancer Institute 2nd Annual Conference, London, UK. Jaishree Bhosle Jenny Wu, Andreas Makris, John A Hartley and Daniel Hochhauser. Modulation of topoisomerase IIα poison induced DNA damage and repair by tyrosine kinase inhibitors. Poster presentation Proceedings of the 100th Annual Meeting of the American Association for Cancer Research; 2009 Apr 18-22. Denver, Colorado, USA AACR 2009; Ab. No. 1829 (Presented by D Hochhauser). Jaishree Bhosle, Andreas Makris, John A Hartley and Daniel Hochhauser. Effect of Chronic exposure to tyrosine kinase inhibitors on chemotherapy-induced DNA damage in the SKBr-3 breast cancer cell line. Poster Presentation San Antonio Breast Cancer Symposium 10-14 Dec 2008, San Antonio, Texas, USA. PUBLICATIONS Boone JJ, Bhosle J, Tilby MJ, Hartley JA, Hochhauser D (2009): Involvement of the HER2 pathway in repair of DNA damage produced by chemotherapeutic agents. Mol Cancer Ther 8: 3015-23. 5 TABLE OF CONTENTS Title page 1 Declaration 2 Abstract 3 Acknowledgments 4 Communications 5 Table of contents 6 Index of figures 22 Index of tables 26 Abbreviations 28 Chapter 1 REVIEW OF THE LITERATURE 33 1.1 Cancer 33 1.2 Breast cancer 35 1.2.1 Presentation of breast cancer 35 1.2.2 Diagnosis of breast cancer 35 1.2.2.1 Pathology of breast cancer 35 1.2.2.2 Staging of invasive breast cancer 37 1.2.3 Prognostic features of invasive breast 37 cancer 1.2.3.1 Pathological features and prognosis 37 1.2.3.2 Genetic profiling in breast cancer 38 1.2.4 Treatment of invasive breast cancer 39 1.2.4.1 Treatment of early invasive breast cancer 39 1.2.4.2 Treatment of metastatic breast cancer 44 1.2.5 Future directions in the treatment of breast 45 cancer 1.3 Human epidermal growth factor receptor family 46 1.3.1 Human epidermal growth factor receptor 46 structure 1.3.2 Activation of human epidermal growth 46 factor receptors 6 1.3.2.1 Ligand dependent activation 47 1.3.2.2 Ligand-independent activation of HER 49 receptors 1.3.2.3 Human epidermal growth factor receptor 49 dimers 1.3.3 Downstream signalling of human epidermal 49 growth factor receptors 1.3.3.1 Phosphatidylinositol 3-kinases signalling 50 1.3.3.2 Ras-Raf-MAPK pathway 51 1.3.3.3 Signal transducers and activators of 51 transcription 1.3.3.4 Receptor internalisation 51 1.3.3.5 Nuclear translocation of human epidermal 52 growth factor receptors 1.3.3.6 Glucose transport 53 1.4 Human epidermal receptor expression and cancer 54 1.4.1 Overexpression of human epidermal 54 receptors 1.4.1.1 EGFR overexpression 54 1.4.1.2 HER2 overexpression 54 1.4.1.3 HER3 overexpression 55 1.4.1.4 HER4 overexpression 55 1.4.2 Increase in the secretion of human 55 epidermal growth factor receptor ligands 1.4.3 Constitutive activating mutations 56 1.4.4 Regulation of human epidermal growth 57 factor receptors by MIG6 1.5 Anti-human epidermal receptor targeted therapies 57 1.5.1 Clinical HER targeted monoclonal antibodies 58 1.5.1.1 Cetuximab (C225/Erbitux®) 58 1.5.1.2 Panitumumab (ABX-EGF/Vectibix®) 59 7 1.5.1.3 Trastuzumab (Herceptin®) 59 1.5.2 Clinical HER targeted small molecule 60 tyrosine kinase inhibitors 1.5.2.1 Gefitinib (ZD 1839/Iressa®) 60 1.5.2.2 Erlotinib (OSI-774/Tarceva®) 60 1.5.2.3. Lapatinib (Tykerb®) 61 1.5.3 Differences between monoclonal antibodies 61 and small molecule tyrosine kinase inhibitor on HER inhibition 1.6 DNA damaging agents in anti-cancer therapy 62 1.6.1 Cisplatin 63 1.6.2 Topoisomerase II poisons 64 1.6.2.1 Isoforms of the topoisomerase II enzyme 64 1.6.2.2 Topoisomerase II function 64 1.6.2.3 Topoisomerase structure 65 1.6.2.4 Targeting topoisomerase II 66 1.6.3 Ionising radiation 67 1.7 Resistance to DNA damaging agents 68 1.7.1 Reduced intracellular drug concentration 68 1.7.2 Conjugation to glutathione 69 1.7.3 Modulation of drug targets 69 1.7.3.1 Resistance to topoisomerase II poisons by 69 reduction in the expression of topoisomerase II 1.7.3.2 Resistance to topoisomerase II poisons by 70 alteration in the location of topoisomerase II 1.7.3.3 Resistance to topoisomerase II poisons by 70 alteration in topoisomerase Ii activity 1.7.4 Cell cycle 71 1.7.5 DNA repair 72 8 1.7.5.1 Repair of cisplatin-induced DNA damage 74 1.7.5.2 Repair of ionising radiation induced DNA 75 damage 1.7.5.3 Repair of topoisomerase II poison induced 76 DNA damage 1.8 Mechanisms of enhancement of cytotoxicity by small 77 molecule tyrosine kinase inhibitors 1.8.1 Inhibition of EGFR activation by tyrosine 78 kinase inhibition 1.8.2 Inhibition of multi-drug resistance 78 transporters by tyrosine kinase inhibitors 1.8.3 Inhibition of conjugation to glutathione by 79 tyrosine kinase inhibitors 1.8.4 Increasing sensitivity to 5-fluorouracil by 79 tyrosine kinase inhibitors 1.8.5 Inhibition of DNA repair by tyrosine kinase 80 inhibitors 1.8.5.1 Inhibition of PI3K/AKT pathway by tyrosine 80 kinase inhibitors 1.8.5.2 Inhibition of nuclear translocation of EGFR 81 and HER2 and inhibition of DNA repair 1.8.5.3 Modulation of DNA-activity and location 81 1.9 Combinations of anti-HER tyrosine kinase inhibitors 81 with chemotherapy 1.9.1 Breast cancer 81 1.9.2 Pancreatic cancer 83 1.9.3 Lung cancer 83 1.9.4 Head and neck cancer 84 1.9.5 Colorectal cancer 84 1.9.6 Ongoing Phase III studies combining 85 lapatinib with chemotherapy 9 1.10 Reasons for failure of tyrosine kinase inhibitors in 85 combination with DNA damaging agents 1.10.1 Failure to inhibit
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