Drug-Resistance and Protective Factors in NSCLC
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Drug-Resistance and Protective Factors in NSCLC Marc Kevin McGowan The Department of Cancer Genetics Institute for Cancer Research The Norwegian Radium Hospital Oslo University Hospital Faculty of Medicine University of Oslo © Marc Kevin McGowan, 2019 Series of dissertations submitted to the Faculty of Medicine, University of Oslo ISBN 978-82-8377-366-8 All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission. Cover: Hanne Baadsgaard Utigard. Print production: Reprosentralen, University of Oslo. Contents Aims of the study ...................................................................................................................... iv Abbreviations ............................................................................................................................. v Papers ....................................................................................................................................... vii Introduction ................................................................................................................................ 1 Lung cancer statistics and aetiology ...................................................................................... 1 Lung cancer histology sub-types. .......................................................................................... 3 Treatment options for lung cancer ......................................................................................... 5 Part I – Potential curative treatments (stages I-III) ............................................................ 5 Part II – Treatment of advanced (stage IV) lung cancers .................................................. 6 Part III – Treatment of EGFR-mutated tumours ................................................................ 6 NSCLC oncogenes ................................................................................................................. 9 Mechanisms of drug-resistance............................................................................................ 14 Yes-associated protein ......................................................................................................... 16 PI3K variants ....................................................................................................................... 19 Cell invasion and EMT ........................................................................................................ 20 Patient selection and cell line models .................................................................................. 21 Materials and methods ............................................................................................................. 23 Cell lines and reagents ......................................................................................................... 23 Cell culture and the generation of drug resistant sub-lines .................................................. 24 Cell viability assay ............................................................................................................... 25 Western Blot ........................................................................................................................ 27 Immunocytochemistry staining ............................................................................................ 29 YAP siRNA knockdown for drug-combination treatment .................................................. 30 RT-qPCR.............................................................................................................................. 31 Tyrosine Kinase Phosphorylation array ............................................................................... 32 Wound heal and doubling time assays ................................................................................. 33 Patient biopsy staining and gene analysis ............................................................................ 34 Survival and prognosis statistics .......................................................................................... 34 Ethics approvals ................................................................................................................... 35 Summary of individual papers ................................................................................................. 36 Paper I .................................................................................................................................. 36 Paper II ................................................................................................................................. 36 i Paper III ............................................................................................................................... 37 Paper IV ............................................................................................................................... 38 Discussion ................................................................................................................................ 39 Methods considerations ....................................................................................................... 39 PIK3CA Patient material .................................................................................................. 39 The choice of cell lines and generation of drug-resistant sub-lines. ................................ 39 Protein identification ........................................................................................................ 40 siRNA targeting YAP over experimental drug inhibition. .............................................. 40 Wound healing and doubling time ................................................................................... 41 Patient biopsy selection for YAP in early stage lung cancers. ........................................ 41 Results discussion .................................................................................................................... 42 Prognostic biomarkers in lung cancer .............................................................................. 42 EGFR TKI resistant sub-line characteristics.................................................................... 43 The role of Yes-associated protein as a mechanism of drug resistance and tumour suppressor ........................................................................................................................ 45 YAP isoform variations and their impact on cancer ........................................................ 49 Potential treatment options for untreatable cancers ......................................................... 50 Further work............................................................................................................................. 52 Conclusion ............................................................................................................................... 53 References ................................................................................................................................ 54 ii Acknowledgments This thesis is the product of hard work, not only from me, but also from the guidance of my supervisors, Dr Odd Terje Brustugun, Dr. Åslaug Helland, and from our team members Dr. Lilach Kleinberg, Dr. Marius Lund-Iversen, Dr. Ann Rita Halvorsen, and Daniel Nebdal for always being on hand with statistics or last minute computational advice. I would also like to thank Dr. Mouldy Sioud for his advice on siRNA transfections and some sneaky agents to get me started, Dr. Else Munthe for her guidance with the live cell imager, and finally Dr. Anne Hansen Ree and Dr. Lise Berven who really came through with the phosphorylation assay and managed to do it without any cost to my project. Thank you for your support and guidance throughout my project. I would like to thank the members of the department of Cancer Research for making me feel welcome. I would like to thank Dr. Xavier Tekpli for making office life more moelluex. Lastly to my family whom have supported me during the difficult time of my last year on the degree. And to my mother, Denise McGowan, who was very proud of my achievements. This thesis is dedicated to her. iii Aims of the study The aims of the thesis were to understand the different mechanisms that can lead to relapse of targeted therapy and activating mutations that may be beneficial for patient survival. The first part of this thesis focused on squamous cell carcinoma and PIK3CA mutations to understand how these mutations may lead to different clinical outcomes in early stage lung cancer. Secondly, to investigate novel mechanisms of drug-resistance in advanced non-small cell lung carcinoma and determine if these are potential therapeutic targets. Therefore the aims were: • Understand how PIK3CA mutations affect patient survival in early stage squamous cell carcinoma in the Norwegian cohort population. • Understand mechanisms in advanced stage drug-resistant lung adenocarcinoma sub- lines with focus on the Yes-associated protein. • Evaluate the Yes-associated protein molecular function differences between drug- resistant sub-lines. • Determine if the Yes-associated protein can be used as a prognostic biomarker of survival in early stage lung cancer patients. iv Abbreviations AC - Adenocarcinoma Akt – Protein kinase B ALK – Anaplastic lymphoma kinase AREG – Amphiregulin Axl – Tyrosine-protein kinase receptor UFO BCA – Bradford coomassie