IDENTIFICATION of a ROLE for INTEGRIN ALPHA 5 in COLONIC EPITHELIAL MORPHOGENESIS by Alina Starchenko Dissertation Submitted To
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IDENTIFICATION OF A ROLE FOR INTEGRIN ALPHA 5 IN COLONIC EPITHELIAL MORPHOGENESIS By Alina Starchenko Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Cell and Developmental Biology May 2017 Nashville, TN Approved: Robert J. Coffey, M.D. Matthew Tyska, Ph.D. Ian Macara, Ph.D. Andreis Zijlstra, Ph.D. Roy Zent, M.D.,Ph.D. To my father, for teaching me how to think To my mother, for teaching me how to learn To my sister, for reminding me that work is not everything To JW, for showing me how to be harder than the science ii Acknowledgements I must first thank Bob Coffey for accepting me into his lab and overseeing my scientific development. The Coffey lab has been a phenomenally supportive environment for me to learn the “hows” of science. The people in the lab, past and present, have truly made this a great experience. It would take too many pages to enumerate, but I would especially like to thank Jeff, Jennie, Galina, Qin and Jim for their technical teachings and assistance. I would like to thank Will, my first bench neighbour and reagent-recipe guru, for the countless protocols and California dome stories. To Michelle, Annie, and Yang: you all taught me so much about science and maybe a little too much about the joys of having a second x chromosome. To Christine and Emily, for forcing me into a gym habit – I look forward to seeing you in Boston. Thank you also to Jarred, Greg, Daniel, Jumpei and Eliot, for the mix of technical advice and happy hour gossip. I would like to acknowledge Bhumi, who has been there to answer all questions and guide my work forward. Thank you to Yuping, who not only taught me to handle a hoard of lab mice, but introduced me to a myriad of delicious and often strange asian treats – I will miss our constant texting. I would like to thank Meghan, who makes everything in the Coffey lab run smoothly. If the Coffey lab was the world, Ramona, you would be holding it on your shoulders like Atlas. Half the projects in this lab would not exist without your ideas, and I would certainly not be where I am today without your guidance and help. I would like to thank my committee, Matt Tyska, Ian Macara, Andreis Zijlstra and Roy Zent. I would especially like to acknowledge Andreis, for his iii help in guiding the early phases of my time here, and Roy, for his initial skepticism and suggestion of the experiments that ultimately opened the door to some interesting biology and ultimately my thesis project. I would also like to thank Jim Patton and Steve Hann, for all of their advice and hard work to make these programs run smoothly. To my former mentor, Cynthia Reinhart- King: your training enabled me to do the work that I have done here, and I would not have even considered joining a PhD program without the inspiration you provided. Outside of lab, I would like to thank my family, for climbing both metaphorical and literal mountains with me. To my Russian family, Liza and Alicia: you are terrible at visiting but you still manage to be there for me, and I am so grateful for that. To my Nashville family, Diana, Cheryl and Courtney: I could not have asked for better partners in science and crime, and I am excited to see where the future takes all of us. iv Table of Contents Page ACKNOWLEDGEMENTS ................................................................................ iii LIST OF FIGURES .......................................................................................... vi LIST OF ABBREVIATIONS AND ACRONYMS ............................................. viii Chapter I: Introduction .................................................................................................. 1 I.1 Cell polarity ......................................................................................... 3 I.2 Integrins: Overview of structure and signaling .................................. 15 I.3 EGFR as a model receptor tyrosine kinase ...................................... 31 I.4 Colorectal cancer .............................................................................. 37 I.5 Rationale for thesis work .................................................................. 40 II: Characterization of the interaction between EGFR and Integrin β1 in colorectal cancer ...................................................................................... 43 II.1 Introduction ...................................................................................... 43 II.2 Results ............................................................................................ 44 II.3 Discussion ....................................................................................... 49 III: Clustering of Integrin α5 at the lateral membrane restores epithelial polarity in invasive colorectal cancer cells ................................ 58 III.1 Introduction ..................................................................................... 58 III.2 Results ........................................................................................... 59 III.3 Discussion ...................................................................................... 70 IV: Integrin β1 clustering dampens morphologic response of colonic epithelial cells to RTK ligands ..................................................... 98 IV.1 Introduction .................................................................................... 98 IV.2 Results ......................................................................................... 100 IV.3 Discussion .................................................................................... 109 V: Conclusions and future directions ........................................................... 122 V.1 Overview ....................................................................................... 122 V.2 Future directions ........................................................................... 125 VI: Materials and Methods ........................................................................... 137 REFERENCES ............................................................................................ 144 v List of Figures Figure Page I-1: Cell polarity is an essential feature of all cells. .......................................... 2 I-2: Overview of organization and junctions in a simple columnar epithelium. ....................................................................................................... 5 I-3: Schematic of polarity protein signaling that drives epithelial polarization. ..................................................................................................... 9 I-4: Integrin structure determines ligand binding ability. ................................. 17 I-5: A schematic of integrin activation and signalling. .................................... 20 I-6: Overview of EGFR family members and signaling ................................. 34 I-7: Aberrant EGFR signaling acts as a driver of malignancy. ....................... 36 I-8: Overview of colonic crypt structure and CRC progression ...................... 39 II-1: Fibrillar type-1 collagen organization changes during colorectal cancer progression. ....................................................................................... 52 II-2: Interaction between EGFR and integrin β1 is increased in colorectal cancer. .......................................................................................... 53 II-3: Interaction between EGFR and integrin β1 is increased in colorectal cancer. .......................................................................................... 54 II-4: CC/SC 3D model system recapitulates in vivo observations in vitro. ...................................................................................... 55 II-5: EGFR ligands AREG and EGF elicit different phenotypic effects on CC cells in 3D. .......................................................................................... 56 II-6: Treatment with AREG but not EGF increases abundance of EGFR/integrin β1 interaction in 2D. ............................................................... 57 III-1: Integrin β1-activating mAb P4G11 abrogates invasive phenotype in colorectal cancer (CRC) cells in 3D. .......................................................... 76 III-2: P4G11 restores apico-basolateral polarity and epithelial cell-cell junctions in 3D. .............................................................................................. 77 III-3: Differential processing of Integrin β1 by two Integrin β1-activating mAbs. ...................................................................................... 78 III-4: P4G11 clustering of Integrin β1 is necessary for rescue of epithelial cyst architecture in SC cells grown in 3D type-1 collagen. ............. 79 III-5: P4G11 selectively induces membrane localization of integrin α5β1. ................................................................................................ 81 vi III-6: Integrin α5 is necessary for P4G11-mediated restoration of epithelial junctions in vitro. ............................................................................. 83 III-7: Integrin α5 clustering leads to fibronectin polymerization at the lateral surface in vitro. ................................................................................... 84 III-8: Fibronectin is necessary and sufficient to induce re-distribution of integrin α5 and paxillin to the lateral membrane