University of Kentucky UKnowledge Theses and Dissertations--Toxicology and Cancer Biology Toxicology and Cancer Biology 2016 ROLE OF SULFIREDOXIN INTERACTING PROTEINS IN LUNG CANCER DEVELOPMENT Hedy Chawsheen University of Kentucky, [email protected] Digital Object Identifier: http://dx.doi.org/10.13023/ETD.2016.176 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Chawsheen, Hedy, "ROLE OF SULFIREDOXIN INTERACTING PROTEINS IN LUNG CANCER DEVELOPMENT" (2016). Theses and Dissertations--Toxicology and Cancer Biology. 13. https://uknowledge.uky.edu/toxicology_etds/13 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]. 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Hedy Chawsheen, Student Dr. Qiou Wei, Major Professor Dr. Isabel Mellon, Director of Graduate Studies ROLE OF SULFIREDOXIN INTERACTING PROTEINS IN LUNG CANCER DEVELOPMENT 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 Hedy Ahmed Chawsheen Lexington, Kentucky Co-Directors: Dr. Qiou Wei, Assistant Professor of Toxicology and Cancer Biology and : Dr. Daret St. Clair, Professor of Toxicology and Cancer Biology Lexington, Kentucky Copyright © Hedy Chawsheen 2016 ABSTRACT OF DISSERTATION ROLE OF SULFIREDOXIN INTERACTING PROTEINS IN LUNG CANCER DEVELOPMENT Sulfiredoxin (Srx) is an antioxidant enzyme that can be induced by oxidative stress. It promotes oncogenic phenotypes of cell proliferation, colony formation, migration, and metastasis in lung, skin and colon cancers. Srx reduces the overoxidation of 2-cysteine peroxiredoxins in cells, in addition to its role of removing glutathione modification from several proteins. In this study, I explored additional physiological functions of Srx in lung cancer through studying its interacting proteins. Protein disulfide isomerase (PDI) family members, thioredoxin domain containing protein 5 (TXNDC5) and protein disulfide isomerase family A member 6 (PDIA6), were detected to interact with Srx. Therefore, I proposed that TXNDC5 and PDIA6 are important for the oncogenic phenotypes of Srx in lung cancer. In chapter one, I presented background information about the role of Srx as an antioxidant enzyme in cancer. I also explained the functional significance of PDIs as oxidoreductase and chaperones in cells. In chapter two, I verified the Srx- TXNDC5/PDIA6 interaction in HEK293T and A549 cells by co-immunoprecipitation and other assays. In TXNDC5 and PDIA6, the N-terminal thioredoxin-like domain (D1) is determined to be the main platform for interaction with Srx. The Srx-TXNDC5 interaction was enhanced by H2O2 treatment in A549 cells. Srx was determined to localize in the endoplasmic reticulum (ER) of A549 cells along with TXNDC5 and PDIA6. This localization was confirmed by both subcellular fractionation and immunofluorescence imaging experiments. In chapter three I focused on studying the physiological function of Srx interacting proteins in the ER. A549 subcellular fractionation results showed that TXNDC5 facilitates Srx retention in the ER. Moreover, TXNDC5 and Srx were found to participate in chaperone activities in lung cancer. Both proteins contributed in the refolding of heat-shock induced protein aggregates. In addition, TXNDC5 and PDIA6 were found to enhance the protein refolding in response to H2O2 treatment. Conversely, Srx appeared to have an inhibitory effect on protein folding under same treatment conditions. Downregulation of Srx, TXNDC5, or PDIA6 significantly reduced cell viability in response to tunicamycin treatment. TXNDC5 knockdown decreased the time required for the splicing of X-box binding protein-1 (XBP-1). In either knockdown Srx or TXNDC5 cells, there was an observable decrease in the expression of GRP78 and the splicing of spliced XBP-1. These results suggest a possible role of Srx in unfolded protein response signaling. TXNDC5 and PDIA6, similar to Srx, contribute to the proliferation, anchorage independent colony formation and migration of lung cancer cells. In this dissertation I concluded that Srx TXNDC5, and PDIA6 proteins participate in oxidative protein folding in lung cancer. Srx and TXNDC5 can modulate unfolded protein response (UPR) sensor activation and growth inhibition. Furthermore, TXNDC5 and PDIA6 can promote tumorigenesis of lung cancer cells. Therefore, the molecular interaction of Srx with TXNDC5/PDIA6 has the potential to be used as novel therapeutic targets for lung cancer treatment. KEYWORDS: Sulfiredoxin, thioredoxin domain containing protein 5, protein disulfide isomerase A isoform 6, interaction, function Hedy Ahmed Chawsheen April 26, 2016 ROLE OF SULFIREDOXIN INTERACTING PROTEINS IN LUNG CANCER DEVELOPMENT By Hedy Ahmed Chawsheen Qiou Wei, Ph.D. Co-Director of Dissertation Daret St. Clair, Ph.D. Co-Director of Dissertation Isabel Mellon, Ph.D. Director of Graduate Studies April 26, 2016 I dedicate this dissertation to my family ACKNOWLEDGMENTS I want to thank my advisor, Dr. Qiou Wei, for providing me the opportunity to work in his lab. I am grateful for all his contribution of time, guidance and support, to make my research experience productive and inspiring. I am privileged to have Dr. Daret St. Clair, Dr. Tadahide Izumi, Dr. Hsin-Sheng Yang, and Dr. Jia Luo as members in my committee, with much of appreciation for their valuable feedbacks on my research. I would also like to thank Dr. Edmond Rucker, who is the external examiner of the final exam. I would like to acknowledge the Human Capacity Development Program of Ministry of Higher Education and Scientific Research in Kurdistan Regional Government-Iraq for their financial sponsorship during my coursework at the University. I would also like to express my gratitude to Dr. Hong Jiang for her patience and generosity in providing technical assistance when I needed. Thanks also go to my colleague, Murli Mishra, for his help and cooperation in the lab. This work would not be possible without the inspiration from my parents, Ahmed Chawsheen and Sabria Mustafa, and my brothers and sisters. Much of appreciation goes to my husband, Zana R. Majeed, for his constant encouragement and support throughout my study period. iii TABLE OF CONTENTS Acknowledgments ............................................................................................................ iii Table of Contents ............................................................................................................. iv List of Figures ................................................................................................................... vi Chapter One: Introduction 1.1. Background ............................................................................................................ 1 Srx in Cancer ............................................................................................................. 3 The PDI Family .......................................................................................................... 5 TXNDC5 and PDIA6 in Cancer ................................................................................ 15 Research Objectives ................................................................................................ 20 1.2. Materials and Methods ......................................................................................... 21 Cell Lines and Chemicals ........................................................................................ 21 Plasmid Constructs and Lentiviral Production ......................................................... 21 Site Directed Mutagenesis ....................................................................................... 24 Protein Purification ..................................................................................................