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  • INVESTIGATION OF EPITHELIAL CANONICAL AND NON-CANONICAL NF-κB SIGNALING IN

    LUNG ADENOCARCINOMA

    By

    Jamie Ausborn Saxon

    Dissertation

    Submitted to the Faculty of the

    Graduate School of Vanderbilt University

    in partial fulfillment of the requirements

    for the degree of

    DOCTOR OF PHILOSOPHY

    in

    CANCER BIOLOGY

    May, 2016

    Nashville, TN

    Approved:

    Timothy S. Blackwell, M.D.

    Ann Richmond, Ph.D.

    Stephen W. Fesik, Ph.D.

    Barbara Fingleton, Ph.D.

  • ii

    Copyright © 2016 by Jamie Ausborn Saxon All Rights Reserved.

  • iii

    For my husband James, my biggest cheerleader

    In honor of my mom, an example of strength

    In honor of my dad, a model of hard work and integrity

    In memory of Roland and Patricia Ausborn and Armand and Mager DeSollar

  • iv

    ACKNOWLEDGEMENTS

    I would first like to acknowledge my advisor, Tim Blackwell, for his guidance throughout my

    graduate school career and for giving me the space and support to develop into an independent scientist.

    In addition, I would like to thank my committee members, Ann Richmond, Stephen Fesik, Barbara

    Fingleton, and William Pao, for their invaluable advice and insights regarding both my project and my

    career. I am grateful for my colleagues in the Cancer Biology Department and the Pulmonary Division as

    well as the members of the Blackwell lab group, past and present, for their input and contributions to this

    work and my training. Particularly, I would like to acknowledge Dong-Sheng Cheng, Wei Han, Linda

    Gleaves, and Rasul Abdolrasulnia for always being available and willing to answer my questions or to

    help me search for the right answers, Jiqing Sai and Hui Yu for sharing their areas of expertise which

    truly enhanced this work, Dawn Newcomb Baker and Tammy Sobolik for helpful feedback on everything

    from qualifying exam proposals to presentations to experiments, and Fiona Yull and Whitney Barham for

    their enthusiasm and eagerness to contribute both ideas and resources. I am also especially grateful for

    Allyson McLoed Perry, my Blackwell lab partner in crime, who has been a great friend and colleague,

    and my graduate school classmates Hayley Clay, Christi French, Sudipta Chakraborty, David Cappel,

    Bobby Madamanchi, Clint Hasenour, Chris Muller, Scott Collier, Sarah Collier, and Seema Sinha, who

    have shared in the struggles and successes of graduate school alongside me.

    Personally, I would like to acknowledge my parents, Chris and Pat Ausborn, for teaching me the

    value of education and always allowing me to pursue opportunity, and my brother Wyatt, who has

    supported and encouraged me in all of my endeavors. Last but not least, I am grateful for my husband

    James, who challenges me to always strive for more and with whom I am lucky to share this adventure.

    This work was supported by the Department of Veterans Affairs Merit Review Award

    1101BX002378 and NIH grants HL092870 and HL085317.

  • v

    TABLE OF CONTENTS

    Page

    DEDICATION…...………………......………............................................................................................iii ACKNOWLEDGEMENTS ......................................................................................................................... iv LIST OF TABLES ..................................................................................................................................... viii LIST OF FIGURES ..................................................................................................................................... ix LIST OF ABBREVIATIONS ...................................................................................................................... xi Chapter I: Introduction .............................................................................................................................................. 1

    Lung Cancer ..................................................................................................................................... 1 Lung Adenocarcinoma ..................................................................................................................... 2 Inflammation in Cancer ................................................................................................................... 5 NF-κB Signaling ............................................................................................................................ 10 Canonical NF-κB Signaling in Cancer .......................................................................................... 13 Non-Canonical NF-κB Signaling in Cancer .................................................................................. 14 Summary and Dissertation Goals ................................................................................................... 16

    II: Epithelial canonical NF-κB signaling promotes EGFR-driven lung carcinogenesis via macrophage recruitment .................................................................................................................................................. 19

    Rationale ........................................................................................................................................ 19 Materials and Methods ................................................................................................................... 20

    Animal models ........................................................................................................................ 20 Tumor histological analysis .................................................................................................... 21 Immunostaining ...................................................................................................................... 22 CT imaging ............................................................................................................................. 22 RNA isolation, RT-PCR, and quantitative real-time PCR ...................................................... 22 Cell lines ................................................................................................................................. 23 Western blot analysis .............................................................................................................. 23 Flow cytometry ....................................................................................................................... 23 In vitro migration assays ......................................................................................................... 24 Neutrophil depletion................................................................................................................ 25 Clodronate macrophage depletion ........................................................................................... 25 Protein array ............................................................................................................................ 25 Enzyme-linked immunosorbent assays (ELISAs) ................................................................... 26 Statistics .................................................................................................................................. 26

    Results ............................................................................................................................................ 26 Inhibition of epithelial canonical NF-κB signaling reduces EGFRL858R-mediated lung

    tumorigenesis .................................................................................................................... 26

  • vi

    Epithelial canonical NF-κB inhibition does not affect epithelial cell survival or activation of EGFR-regulated signaling pathways ............................................................................ 27

    Canonical NF-κB inhibition reduces macrophage and neutrophil influx into the lungs of mice expressing mutant EGFR ......................................................................................... 29

    Canonical NF-κB inhibition decreases lung tumors in mice expressing a TKI-resistant EGFR mutation ................................................................................................................. 31

    Canonical NF-κB signaling promotes macrophage and neutrophil chemotaxis ..................... 33 Macrophages promote EGFR-mediated lung tumorigenesis .................................................. 35

    Discussion ...................................................................................................................................... 37 III: Non-canonical NF-κB signaling resulting from p52 expression enhances lung cancer progression through regulation of cell cycle genes ........................................................................................................ 41

    Rationale ........................................................................................................................................ 41 Materials and Methods ................................................................................................................... 43

    Generation of CCSP-p52 mouse model ..................................................................................

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