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Determining Mechanisms of Response to Polo-Like Kinase 1 Inhibition in Non-Small Cell Lung Cancer

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Determining Mechanisms of Response to Polo-Like Kinase 1 Inhibition in Non-Small Cell Lung Cancer The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 8-2016 Determining Mechanisms of Response to Polo-like Kinase 1 Inhibition in Non-Small Cell Lung Cancer Ruchitha Goonatilake Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Medicine and Health Sciences Commons Recommended Citation Goonatilake, Ruchitha, "Determining Mechanisms of Response to Polo-like Kinase 1 Inhibition in Non- Small Cell Lung Cancer" (2016). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 700. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/700 This Thesis (MS) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. DETERMINING MECHANISMS OF RESPONSE TO POLO-LIKE KINASE 1 INHIBITION IN NON-SMALL CELL LUNG CANCER by Ruchitha Goonatilake, BS APPROVED: ______________________________ Faye M. Johnson, MD, Ph.D., Advisory Professor ______________________________ Khandan Keyomarsi, Ph.D. ______________________________ Walter N. Hittelman, Ph.D. ______________________________ Lauren A. Byers, MD. ______________________________ Jing Wang, Ph.D. APPROVED: ____________________________ Dean, The University of Texas Graduate School of Biomedical Sciences at Houston iv DETERMINING MECHANISMS OF RESPONSE TO POLO-LIKE KINASE 1 INHIBITION IN NON-SMALL CELL LUNG CANCER A THESIS Presented to the Faculty of The University of Texas Health Science Center at Houston and The University of Texas MD Anderson Cancer Center Graduate School of Biomedical Sciences in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE by Ruchitha Goonatilake, BS Houston, Texas Date of Graduation August 2016 5 Dedicated to my family 6 Acknowledgements: I would like to express my sincere gratitude to my mentor Dr. Faye Johnson for her kindness, patience, and support while she gave me guidance for the past two years. She was a fantastic mentor and I aspire to be bit like her one day. I am also very grateful for the help I received from all the members of our lab in so many ways: Dr. Tuhina Mazumdar, Dr. Shaohua Peng, Dr. Nene Kalu, Dr. Ratnakar Singh, Dr. Vaishnavi Sambandam, Dr. Ming Zhang, Dr. Yuehong Wang, and Liguang Wang. I am also very grateful to Dr. Uma Giri who has given me a great deal of advice through my time here. I would also like to sincerely thank my committee members Dr. Khandan Keyomarsi, Dr. Walter Hittelman, Dr. Lauren Byers, and Dr. Jing Wang for their excellent advice given during committee meetings. Additionally, I would like to thank the Bioinformatics department who worked very hard to help us find significant results which we studied further in the laboratory. Without them, my life would have been much more difficult. Also, I would like to thank David Peng and Dr. Don Gibbons for their collaboration with regards to the EMT experiments, and Dr. Jonathan Roybal for teaching me to use the fluorescent microscope. Finally, a big thank you to the flow cytometry core for teaching me to run a flow cytometer. 7 Determining Mec hanisms of Response to Polo-like Kinase 1 Inhibition in Non-Small Cell Lung Cancer Ruchitha Goonatilake, B.S. Supervisory Professor: Faye M. Johnson, M.D., Ph.D. Non-small c ell l ung c ancer ( NSCLC) i s the l eading c ause o f c ancer-related deat h worldwide. The discovery of genetic alterations in some patients (~15%) has made it possible to use targeted therapies without the use of chemotherapy. To identify potential therapeutic targets in NSCLC, we systematically evaluated two cancer cell line databases with sensitivity data for a broad range of drugs. We identified polo-like kinase 1 (Plk1) as the most promising target for further i nvestigation ba sed on a s ubset o f s ensitive c ell l ines and i nhibitors t hat w ere i n advanced clinical development. To identify potential biomarkers of response and mechanisms of Plk1 inhibitor- induced apoptosis, we performed an i ntegrated analysis of gene and pr otein expression, gene mutation, and dr ug sensitivity using three Plk1 inhibitors (volasertib, BI2536, GSK461364) in a large panel o f N SCLC c ell l ines. We obs erved t hat t he N SCLC c ell l ines ha ve v arying sensitivities t o P lk1 in hibition, w ith a s maller subset de monstrating s ensitivity t o al l t hree inhibitors. Plk1 inhibition led to increase of cells with 4N DNA content, but only sensitive cell lines underwent substantial apoptosis following Plk1 inhibition. NSCLC lines with a high epithelial-mesenchymal t ransition gene signature s core (i.e., mesenchymal l ines) w ere m ore sensitive to Plk1 inhibitors than epithelial lines (p<0.02). Similarly, proteomic profiling demonstrated that E-cadherin expression was higher in the resistant cell lines (p<0.01). Induction o f an epi thelial phenot ype us ing miR-200 ex pression i ncreased r esistance t o P lk1 inhibition, w hereas ex pression o f ZE B1 i ncreased s ensitivity t o P lk1 i nhibition. A dditionally, treatment o f with TGF-β to induce EMT sensitized a resistant cell line and led to increased 8 apoptosis a fter P lk1 i nhibition, c ompared t o par ental c ells. KRAS mutation and a lterations i n tight-junction, ErbB, and Rho signaling pathways also correlated with drug response. We dem onstrate t hat epi thelial-mesenchymal t ransition l eads to P lk1 i nhibitor sensitivity in a la rge-scale, i ntegrated anal ysis o f P lk1 i nhibitor sensitivity. O ur findings hav e important clinical implications for mesenchymal NSCLC, as a s ignificant subset of this disease associated with resistance to currently approved targeted therapies. 9 TABLE OF CONTENTS: Approval Sheet..........................................................................................................3 Title Page..................................................................................................................4 Dedication................................................................................................................5 Acknowledgements.................................................................................................6 Abstract.....................................................................................................................7 Table of Contents.....................................................................................................9 List of Figures..........................................................................................................13 List of Tables............................................................................................................15 Chapter 1. Introduction............................................................................................16 Lung Cancer Background……………………………………...........................17 Non-small cell lung cancer…………………………….....................................17 Common genetic aberrations in NSCLC.......................................................18 Treatment of NSCLC.......................................................................................18 Hallmarks of cancer…………………………....................................................19 Phases of the cell cycle………………………………………………………......20 Cyclins, CDKs, and Mitotic Entry…………………..………………………......22 Plk family…....................................................................................................23 Plk1 regulation...............................................................................................26 Plk1 and mitotic entry....................................................................................27 Plk1 and DNA damage response..................................................................28 Plk1 and the spindle assembly checkpoint.................................................29 Plk1 in cancer.................................................................................................30 10 Plk1 inhibitors in cancer................................................................................30 Plk1 inhibitors in NSCLC...............................................................................33 Epithelial to Mesenchymal Transition.........................................................33 EMT and cancer.............................................................................................35 EMT and cell cycle.........................................................................................36 Chapter 2. Materials and Methods........................................................................38 Antibodies and reagents............................................................................39 Cell culture and characterization...............................................................39 Cell viability assays…….............................................................................40 Calculation of the drugs’ effects on viability............................................40
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