University of Kentucky UKnowledge Theses and Dissertations--Toxicology and Cancer Biology Toxicology and Cancer Biology 2020 THE ROLE OF NEURAL PRECURSOR CELL EXPRESSED DEVELOPMENTALLY DOWN-REGULATED PROTEIN 9 IN ENHANCED AGGRESSIVENESS OF HEXAVALENT CHROMIUM TRANSFORMED BRONCHIAL EPITHELIAL CELLS Peter Van Wie University of Kentucky, [email protected] Digital Object Identifier: https://doi.org/10.13023/etd.2020.041 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Van Wie, Peter, "THE ROLE OF NEURAL PRECURSOR CELL EXPRESSED DEVELOPMENTALLY DOWN- REGULATED PROTEIN 9 IN ENHANCED AGGRESSIVENESS OF HEXAVALENT CHROMIUM TRANSFORMED BRONCHIAL EPITHELIAL CELLS" (2020). Theses and Dissertations--Toxicology and Cancer Biology. 30. https://uknowledge.uky.edu/toxicology_etds/30 This Doctoral Dissertation is brought to you for free and open access by the Toxicology and Cancer Biology at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Toxicology and Cancer Biology by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Peter Van Wie, Student Dr. Isabel Mellon, Major Professor Dr. Isabel Mellon, Director of Graduate Studies THE ROLE OF NEURAL PRECURSOR CELL EXPRESSED DEVELOPMENTALLY DOWN-REGULATED PROTEIN 9 IN ENHANCED AGGRESSIVENESS OF HEXAVALENT CHROMIUM TRANSFORMED BRONCHIAL EPITHELIAL CELLS DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Medicine at the University of Kentucky By Peter Gregory Van Wie Lexington, Kentucky Director: Dr. Isabel Mellon, Professor of Toxicology and Cancer Biology Lexington, Kentucky 2019 Copyright © Peter Van Wie 2019 ABSTRACT OF DISSERTATION THE ROLE OF NEURAL PRECURSOR CELL EXPRESSED DEVELOPMENTALLY DOWN-REGULATED PROTEIN 9 IN INHANCED AGGRESSIVENESS OF HEXAVALENT CHROMIUM TRANSFORMED BRONCHIAL EPITHELIAL CELLS Hexavalent chromium (Cr(VI)) is classified as a confirmed human carcinogen by the International Agency for Research and Cancer (IARC) and by the U.S. Environmental Protection Agency (EPA). Chronic exposure to (Cr(VI)) causes malignant cell transformation in human bronchial epithelial BEAS-2B cells. These Cr(VI)-transformed cells exhibit a highly aggressive phenotype including increased migration, invasion, and angiogenesis. The Cas family protein neuronal precursor developmentally down regulated protein 9 (NEDD9/Cas-L/HEF1) was dramatically overexpressed in Cr(VI)-transformed cells compared to normal BEAS-2B cells. Knockdown of NEDD9 by its shRNA reduced migration and invasion in vitro measured by migration and invasion assays. shNEDD9 reduced tumor formation in nude mice injected subcutaneously and metastasis in mice injected in the lateral tail vein. NEDD9 is critical for the activation of c-Src kinase (Src) at Y416 and overexpression of focal adhesion kinase (FAK). These interactions with NEDD9, FAK, and Src indicate that NEDD9 was acting upstream from FAK and Src in Cr(VI)- transformed cells. NEDD9 expression or Src activation had downstream effects on migration and invasion. Interestingly β-Catenin activity was positively regulated by the expression of NEDD9 and the activation of Src. NEDD9 is critical for HIF-1α expression which leads to angiogenesis. The NEDD9 mediated angiogenic proteins VEGF, PECAM, ANG1, and MMP proteins are overexpressed in Cr(VI)-transformed cells. NEDD9 may increase angiogenesis by first mediating the phosphorylation of p38 and then HIF-1α. NEDD9 can also bind directly to CREB binding protein (CBP) a critical cofactor for HIF- 1α transcriptional activity. These results lead us to believe that NEDD9 is a driving force in lung cancer aggressiveness by magnifying migration and invasion by facilitating FAK/Src binding and activation of Src kinase, by increasing β-Catenin activity and by increasing angiogenesis through HIF-1α stabilization and overexpression of downstream angiogenic proteins. KEYWORDS: NEDD9, Chromium(VI), FAK, Src, β-Catenin, HIF-1α Peter Gregory Van Wie (Name of Student) 12/20/2019 Date THE ROLE OF NEURAL PRECURSOR CELL EXPRESSED DEVELOPMENTALLY DOWN-REGULATED PROTEIN 9 IN INHANCED AGGRESSIVENESS OF HEXAVALENT CHROMIUM TRANSFORMED BRONCHIAL EPITHELIAL CELLS By Peter Gregory Van Wie Dr. Isabel Mellon Director of Dissertation Dr. Isabel Mellon Director of Graduate Studies 12/20/2019 Date TABLE OF CONTENTS LIST OF FIGURES ...................................................................................................................................... vi CHAPTER 1. Introduction to environmental and occupational exposure to hexavalent chromium and tumor aggressiveness ............................................................................................................................... 1 1.1 Exposure to Heavy Metal .................................................................................................................. 1 1.2 Tumorigenesis ................................................................................................................................... 4 1.3 Aggressiveness .................................................................................................................................. 7 CHAPTER 2. Upregulation of NEDD9 promotes migration and invasion through FAK/Src and β- Catenin/E-Cadherin pathways in Chromium(VI)-transformed bronchial epithelial cells.......................... 13 2.1 Abstract ........................................................................................................................................... 13 2.2 Introduction ..................................................................................................................................... 14 2.3 Methods .......................................................................................................................................... 16 2.3.1 Chemicals and Reagents ........................................................................................................ 16 2.3.2 Cell Culture ........................................................................................................................... 16 2.3.3 Stable NEDD9 knockdown cells ........................................................................................... 17 2.3.4 Immunoblotting analysis ....................................................................................................... 17 2.3.5 Immunoprecipitation ............................................................................................................. 17 2.3.6 Real time PCR ....................................................................................................................... 17 2.3.7 Migration and Invasion assays............................................................................................... 18 2.3.8 ELISA assay .......................................................................................................................... 18 2.3.9 Kinase activity assay ............................................................................................................. 18 2.3.10 In vivo tail vein injection assay ......................................................................................... 18 2.3.11 Statistical analysis ............................................................................................................. 18 2.4 Results ............................................................................................................................................. 19 2.4.1 Upregulation of NEDD9 in Cr(VI)-transformed BEAS-2B cells .......................................... 19 2.4.2 Knockdown of NEDD9 reduces migration and invasion in Cr(VI)-transformed cells .......... 19 2.4.3 Knockdown of NEDD9 reduces metastasis in vivo ............................................................... 20 2.4.4 Upregulation of NEDD9 increases aggressiveness by facilitating