University of Louisville ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations 5-2019 Paraoxonase 2 is critical for non-small cell lung carcinoma proliferation. Aaron Whitt University of Louisville Follow this and additional works at: https://ir.library.louisville.edu/etd Part of the Cancer Biology Commons, Molecular Biology Commons, and the Pharmacology Commons Recommended Citation Whitt, Aaron, "Paraoxonase 2 is critical for non-small cell lung carcinoma proliferation." (2019). Electronic Theses and Dissertations. Paper 3236. https://doi.org/10.18297/etd/3236 This Master's Thesis is brought to you for free and open access by ThinkIR: The nivU ersity of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The nivU ersity of Louisville's Institutional Repository. This title appears here courtesy of the author, who has retained all other copyrights. For more information, please contact [email protected]. PARAOXONASE 2 IS CRITICAL FOR NON-SMALL CELL LUNG CARCINOMA PROLIFERATION By Aaron Whitt B.S., Morehead State University, 2010 A Thesis Submitted to the Faculty of the School of Medicine of the University of Louisville in Partial Fulfillment of the Requirements for the Degree of Master of Science in Pharmacology and Toxicology Department of Pharmacology and Toxicology University of Louisville Louisville, Kentucky May, 2019 PARAOXONASE 2 IS CRITICAL FOR NON-SMALL CELL LUNG CARCINOMA PROLIFERATION By Aaron Gregory Whitt B.S., Morehead State University, 2010 A Thesis Approved on December 13, 2018 By the following Thesis Committee _______________________________ Chi Li, Ph. D. _______________________________ Levi Beverly, Ph. D. _______________________________ Geoffrey Clark, Ph. D. _______________________________ Brian Clem, Ph. D. _______________________________ Robert Mitchell, Ph. D. ii ACKNOWLEDGMENTS First, I would like to thank my mentor, Dr. Chi Li, who has guided, through instruction, and inspired, through demonstration, my personal journey as a scientist. His work ethic and technical mastery are unparalleled. My gratitude is extended to members of my committee—Drs. Levi Beverly, Geoffrey Clark, Brian Clem, and Robert Mitchell—for lending their expertise to fortify current and future research endeavors. To current and former members of the Li laboratory— Guoping, Aaron, Ellen, and Jane—who are endlessly willing to answer a question or lend a hand. To my fiancée, Kelly, for her unwavering love and support through all of life’s hurdles. Finally, to my family, for their love and encouragement. iii ABSTRACT PARAOXONASE 2 IS CRITICAL FOR NON-SMALL CELL LUNG CARCINOMA PROLIFERATION Aaron Whitt December 13, 2018 Non-small cell lung carcinoma (NSCLC) comprises 85% of lung cancer diagnoses and is plagued by drug resistance. Thus, elucidating the underlying mechanisms of NSCLC is paramount to expand future treatment options. Paraoxonase 2 (PON2), an intracellular enzyme with arylesterase and lactonase functions, has well-established anti-atherosclerotic activity. Recent studies show PON2 is overexpressed in a variety of tumors and confers drug resistance, although these interactions have not been thoroughly examined in NSCLC. Thus, we sought to investigate the role of PON2 in cellular proliferation using PON2- knockout mice, primary mouse cells, and NSCLC cell lines. Using these approaches, we demonstrate that PON2 is required for NSCLC proliferation, but dispensable for normal mouse development and non-transformed cell proliferation. These observations highlight PON2 as a potential therapeutic target against NSCLC. iv TABLE OF CONTENTS SECTION PAGE ACKNOWLEDGMENTS iii ABSTRACT iv LIST OF FIGURES vi INTRODUCTION 1 RESEARCH OBJECTIVE 12 METHODS 13 RESULTS 23 DISCUSSION 44 CONCLUSION 48 REFERENCES 49 CURRICULUM VITA 55 v LIST OF FIGURES FIGURE PAGE 1. PON2 is upregulated in NSCLC tumor samples 25 2. Generation of PON2-knockout mice using a CRISPR/Cas9 27 approach 3. Tissues from PON2-knockout mice lack PON2 expression 29 4. Primary MEFs are sensitive to C12-mediated cell death in a 31 PON2-dependent manner 5. PON2 enzymatic activity is abrogated in tissues and primary 34 cells from PON2-knockout mice 6. Normal mouse growth and development is unaffected by PON2 36 deficiency 7. PON2 deficiency hinders proliferation of NSCLC cells 39 8. PON2 knockdown fails to affect untransformed human epithelial 41 cell proliferation 9. Deficiency in PON2 expression induces G1 cell cycle arrest of 43 A549 cells vi CHAPTER 1: INTRODUCTION 1. Paraoxonases The paraoxonases (PONs) comprise 3 members (PON1, PON2, and PON3) of an antioxidant enzyme family encoded in a gene cluster on the long arm of chromosome 7 (7q21.3-22.1) [1]. These enzymes are evolutionarily ancient, first appearing in invertebrates, and are highly conserved in mammals, displaying 80- 95% sequence identity at both the amino acid and nucleotide level [2]. PON2 is presumed to be the oldest, eventually giving rise to PON1 and PON3 through gene duplication events [3]. All PONs exhibit lactonase and arylesterase activity, but are otherwise functionally diverse [4]. For instance, PON1 was first discovered and named for its ability to hydrolyze paraoxon, the toxic metabolite of the organophosphate insecticide, parathion [5]. Although their name would suggest the contrary, PON2 and PON3 lack this paraoxon-detoxifying activity but retain the designation based on genetic homology. Intriguingly, no endogenous substrates have been identified for this class of enzymes. 1.1 PON1 PON1 is the most characterized of the PONs. Early work on PON1 focused on its xenobiotic metabolizing capability, specifically in the context of organophosphate detoxification, [6], [7]. Recent and emerging PON1 research, 1 however, has shifted to exploring its role in atherosclerosis, immunity, and cancer [8]. The PON1 gene encodes a 43 kDa protein consisting of 354 amino acids [9] which is produced by the liver and released into circulation where it associates with high-density lipoprotein (HDL) particles and is distributed throughout the body [10]. Once in the serum, PON1 inhibits and reverses the oxidization of low-density lipoproteins (LDL), an initiating step in the development of atherosclerosis [11]. This effect has been extensively demonstrated in cell-based systems and in PON1-knockout mice[11]–[14]. Additionally, a number of human studies have suggested a relationship between polymorphisms in the coding and promoter regions of the human PON1 gene and susceptibility to cardiovascular disease [15]. The sheer abundance of research linking PON1 status to atherogenesis has sparked much interest in PON1 as a biomarker and/or therapeutic target against oxidative vascular damage [16]. PON1 has also been shown to mediate bacterial infection. Through its lactonase activity, PON1 can cleave a class of bacterial quorum-sensing compounds, the acyl-homoserine lactones [17]. The hydrolysis of the lactone ring effectively inactivates acyl-homoserine lactones and disrupts bacterial intercellular communication and colonization of host tissues. The antioxidant and anti-inflammatory properties of PON1 have prompted researchers to investigate its role in cancer. Indeed, several published reports confirm a link between PON1 and cancer. An investigation conducted by Marchesani et al. monitored prostate cancer development in a population of Finnish men and found a specific PON1 single nucleotide polymorphism (SNP), 2 I102V, was associated with increased incidence [18]. Similarly, Stevens et al. discovered the L55M SNP correlated to an increased risk of breast cancer in a cohort of post-menopausal women [19]. More recently, Aldonza et al. used data from the cancer genome atlas (TCGA), lung cancer patient samples, and lung cancer cell lines to show that PON1 provides an anti-apoptotic function to stimulate tumor growth and progression [20]. While these observations are promising, the underlying mechanisms remain undetermined. 1.2 PON3 In contrast to PON1, PON3 is the most recently-identified and least-studied member of this family. PON3 is a 40 kDa protein synthesized mainly by the liver and, to a lesser extent, the kidney which associates with circulating plasma HDLs [21]. In circulation, PON3 is capable of detoxifying oxidized LDLs (oxLDLs) at a higher capacity than PON1, although circulating levels of PON3 are much lower than PON1 [21]. PON3 has also been detected intracellularly, localized to endoplasmic reticulum (ER) and mitochondria [22], [23]. Similar to its other family members, PON3 elicits anti-inflammatory, anti-oxidant responses in vitro and in vivo which have been connected to atherosclerosis, diabetes, neurodegenerative disorders, and cancer [2], [5], [24]. 1.3 PON2 PON2, unlike the other PONs, is expressed intracellularly in nearly every tissue surveyed [25], where it localizes to mitochondria, ER, and nuclear lamina 3 [26] as a transmembrane protein with a molecular mass of 43 kDa [27]. PON2 shares anti-oxidant and anti-inflammatory characteristics with other PONs, such as preventing and reversing oxidative damage to LDL [25]. In addition, PON2 also exerts intracellular influence, generally by mitigating oxidative stress in mitochondria and ER. Its widespread tissue distribution and subcellular localization implicate PON2 in a variety of physiological processes 1.3.1 PON2 and Atherosclerosis PON2 is perhaps most thoroughly characterized as a mitigating factor in