SUPPRESSION OF CARCINOGENESIS AND TUMOR PROGRESSION BY AN ENERGY RESTRICTION-MIMETIC AGENT IN MURINE MODELS OF PROSTATE CANCER DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Lisa Danielle Berman-Booty, V.M.D Graduate Program in Veterinary Biosciences The Ohio State University 2013 Dissertation Committee: Professor Ching-Shih Chen, Advisor Professor Robert Brueggemeier Professor Steven Clinton Professor Thomas Rosol Copyrighted by Lisa Danielle Berman-Booty 2013 ABSTRACT Cancer cells preferentially utilize glycolysis to generate energy even in the presence of sufficient oxygen for oxidative phosphorylation. This shift in energy metabolism, termed the Warburg effect, is responsible for cancer cells’ high metabolic rate. Therapies that inhibit cancer cell energy metabolism have proved effective in vitro, and dietary caloric restriction is a valuable experimental chemotherapeutic strategy. While animal studies utilizing caloric restriction typically restrict the experimental group’s caloric intake by 20-40%, this degree of caloric restriction is not realistic for human cancer patients. Therefore, agents that can induce a response similar to that of glucose restriction in vitro and caloric restriction in vivo are needed. These agents are termed energy-restriction mimetic agents (ERMAs) and include 2-deoxyglucose, resveratrol, and the thiazolidinedione derivatives OSU-CG12 and OSU-CG5. Here, we characterized OSU- CG5’s mechanism of action in vitro and its chemotherapeutic and chemopreventive activities in vivo. Specifically, we evaluated OSU-CG5’s activity in three different human prostate cancer cell lines that range from androgen-dependent (LNCaP) to castration- resistant (LNCaP-abl and PC3). OSU-CG5 was cytotoxic and induced a response similar to that of glucose deprivation in these cell lines. Additionally, treatment with OSU-CG5 resulted in decreased expression of genes that promote the Warburg effect, namely, those ii for glucose transporter 1 (GLUT1) and a number of metabolic enzymes. Regarding its in vivo chemotherapeutic activity, 100 mg/kg/day of OSU-CG5 administered via daily oral gavage suppressed the growth of LNCaP-abl xenograft tumors by 81%. Tumor growth suppression was associated with decreased tumor cell proliferation, as determined by proliferating cell nuclear antigen (PCNA) levels, and modulation of intratumoral biomarkers associated with cell survival, growth, and metabolism, including insulin like growth factor 1 (IGF-1) and its receptor IGF-1R, Myc, the androgen receptor (AR), and cyclins D1 and E. OSU-CG5’s activity was also investigated using transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. Treatment of 6-week-old TRAMP mice with 100 mg/kg/day of OSU-CG5 via oral gavage for 4 weeks significantly reduced absolute and relative urogenital tract weights, as well as the weights of the individual lobes of the prostate. Reductions in lobe weight were associated with decreased cell proliferation within prostatic intraepithelial neoplasia (PIN) lesions, as determined by immunostaining for Ki67 and western blotting for PCNA. OSU-CG5 also decreased the levels of the AR, phosphorylated Akt (p-Akt), and IGF-1R within the prostates of these mice. When 6-week-old, intact, male TRAMP mice were fed an AIN- 76A diet containing 1286 ppm of OSU-CG5 for approximately 18 weeks, prostate tumor development was not suppressed or significantly delayed. However, the tumors that developed in OSU-CG5-treated mice were 54.6% smaller by volume and 54.1% smaller by mass than the control mouse tumors. Decreased tumor size was associated with decreased tumor cell proliferation, as determined by Ki67 immunostaining, and reductions in the AR and p-Akt. Collectively, these studies demonstrate OSU-CG5’s iii efficacy as a chemotherapeutic agent. OSU-CG5 mediated this effect by reducing tumor cell proliferation and modulating pro-growth and pro-survival biomarkers. iv DEDICATION This document is dedicated to my husband Jordan and parents Marvin and Ronna v ACKNOWLEDGMENTS I thank my advisor Dr. Ching-Shih Chen for his advice, mentorship and unwavering support. I thank the members of my advisory committee for their guidance. I thank Dr. Samuel Kulp for his advice and help with experimental design, animal studies, and manuscript preparation. I appreciate all the help and encouragement provided by the other members of the Chen lab. In particular, I acknowledge Dr. Po-Chen Chu for his technical expertise and assistance and Dr. Dasheng Wang for synthesizing OSU-CG5. I thank Dr. Jennifer Thomas-Ahner of the Clinton lab for her assistance with mouse necropsies and urogenital tract microdissection. I thank Drs. Brad Bolon and Krista La Perle of the Comparative Pathology and Mouse Phenotyping Shared Resource (CPMPSR) for their guidance and mentorship in mouse and toxicologic pathology. I appreciate Alan Fletchner, Anne Saulsbery, Bill Kimble, and Florinda Jaynes of the CPMPSR for their help with tissue and slide processing and immunohistochemistry. I thank Jody Sneddon of the CPMPSR for performing serum chemistries and complete blood counts. I acknowledge the National Institute of Health for my research support and funding. From June 2009 through April 2012, my research was supported by the T-32 Institutional National Research Service Award (NRSA) in Mouse Models of Human vi Disease (Ruth L. Kirschstein award). From April 2012 through the present time, my research has been supported by a K01 SERCA. Lastly, I thank my husband and parents for their love and support throughout this entire process. vii VITA March 7, 1982 ................................................Born—Brookline, MA, USA 2004 ...............................................................Bachelors of Arts, Biology School of Arts and Sciences University of Pennsylvania Philadelphia, PA 2008 ...............................................................Veterinariae Medicinae Doctoris School of Veterinary Medicine University of Pennsylvania Philadelphia, PA 2008 to present ..............................................Residency in Veterinary Anatomic Pathology Department of Veterinary Biosciences The Ohio State University Columbus, OH 2008 to present ..............................................Postdoctoral Fellow Department of Veterinary Biosciences The Ohio State University Columbus, OH 2012................................................................Diplomate, American College of Veterinary Pathologists (anatomic pathology) viii PUBLICATIONS 1. Berman-Booty LD, Chu PC, Thomas-Ahner JM, Bolon B, Wang D, Yang T, Clinton SK, Kulp SK, Chen CS. Suppression of prostate epithelial proliferation and pro-growth signaling in transgenic mice by a new energy-restriction mimetic agent. Cancer Prev Res in press (PMID: 23275006) 2.Omar HA, Berman-Booty L, Weng JR. Energy restriction: stepping stones towards cancer therapy. Future Oncol 2012; 8 (12): 1503-6 3.Berman-Booty LD, Garzel LM, Bergdall V, La Perle KM. A prostate fibromyxoid sarcoma with smooth muscle differentiation in a F344xBNF1 rat. Vet Pathol 2012; 49(4): 642-647 4.Berman-Booty LD, Sargeant AM, Rosol TJ, Rengel RC, Clinton SK, Chen CS, Kulp SK. A review of the existing grading schemes and a proposal for a modified grading scheme for prostatic lesions in TRAMP mice. Toxicol Pathol 2012; 40(1):5-17 5.Omar HA, Chou CC, Berman-Booty LD, Ma Y, Hung JH, Wang D, Kogure T, Patel T, Terracciano L, Muthusamy N, Byrd JC, Kulp SK, Chen CS. Antitumor effects of OSU- 2S, a non-immunosuppressive analogue of FTY720, in hepatocellular carcinoma. Hepatology 2011; 53(6):1943-58 6.Omar HA, Berman-Booty LD, Kulp SK, Chen CS. Energy restriction as an antitumor target. Future Oncol 2010; 6(11): 1675-9 7.Berman-Booty LD, Cui J, Horvath SJ, Premanandan C. Pathology in Practice. Tularemia. J Am Vet Med Assoc 2010; 237(2): 163-5 8.Zaph C, Troy AE, Taylor BC, Berman-Booty LD, Guild KJ, Du Y, Yost EA, Gruber AD, May MJ, Greten FR, Eckmann L, Karin M, Artis D. Epithelial-cell-intrinsic IKK- beta expression regulates intestinal immune homeostasis. Nature 2007; 446(7135): 552-6 9.Blais MC, Berman L, Oakley D, and Giger U. The canine DAL blood type: a red cell antigen lacking in some Dalmatians. J Vet Intern Med 2007; 21(2): 281-6. ix FIELDS OF STUDY Major Field: Veterinary Biosciences x TABLE OF CONTENTS Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita .................................................................................................................................. viiii List of Tables ................................................................................................................... xiv List of Figures .................................................................................................................. xvi Chapter 1: A review of the existing grading schemes and a proposal for a modified grading scheme for prostatic lesions in TRAMP mice ....................................................... 1 Abstract ........................................................................................................................... 1 Introduction