Targeting Cancer Metabolism with Ketosis and Hyperbaric Oxygen

Transcription

1 University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School January 2014 Targeting Cancer Metabolism with Ketosis and Hyperbaric Oxygen Angela M. Poff University of South Florida, Follow this and additional works at: Part of the Oncology Commons Scholar Commons Citation Poff, Angela M., "Targeting Cancer Metabolism with Ketosis and Hyperbaric Oxygen" (2014). Graduate Theses and Dissertations. This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact

2 Targeting Cancer Metabolism with Ketosis and Hyperbaric Oxygen by Angela Marie Poff A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular Pharmacology and Physiology Morsani College of Medicine University of South Florida Major Professor: Dominic D Agostino, Ph.D. Jay Dean, Ph.D. Patricia Kruk, Ph.D. Marzenna Wiranowska Ph.D. David Diamond, Ph.D. Date of Approval: July 10, 2014 Keywords: Cancer metabolism, Warburg effect, ketone, metastasis Copyright 2014, Angela Marie Poff

3 ACKNOWLEDGMENTS This work was made possible by support from our funding sources, including the Department of Molecular Pharmacology and Physiology, the Office of Naval Research (ONR), and generous charitable contributions from Mike McCandless and Scivation, Inc. I d like to express my sincere appreciation to the many people who have supported me along this journey. First and foremost, to my husband, Franklin Poff, who always enthusiastically supported my goals and dreams, both personal and professional, encouraged me, made me laugh when I was stressed and disheartened, and celebrated my every success, no matter how big or small. To my best friend, soul mate, and partner-in-crime, I am eternally grateful. To Dr. Dominic D Agostino, a wonderful mentor and scientist who has never been anything but supportive and encouraging. I thank him for always making time for me no matter how busy his schedule, for giving me innumerable opportunities to further myself personally and professionally, for always respecting my opinion, and for inspiring me to remember that above all, it is how our work can help patients that matters the most. To the members of my dissertation committee Dr. Jay Dean, Dr. Patricia Kruk, Dr. Marzenna Wiranowska, and Dr. David Diamond for their scientific guidance and support over the past four years. A special thank you is due to Dr. Thomas Seyfried, for taking the time to

4 always provide thoughtful guidance and input on my research and manuscripts, and for teaching me to trust the data above all things. To my lab family, especially Carol Landon, Dr. Jay Dean, Dr. Heather Held, Dr. Raffaele Pilla, Geoffrey Ciarlone, Shannon Kesl, and Nate Ward for making work such an enjoyable and supportive place. To my friends and family who have always supported me personally and professionally, particularly Callan Heath, Frank, Theresa, Will, and Curt Poff, Laura and Dustin Dacus, Jay Neal, and especially my sister and one of my best friends, Christa Neal. To my dad, Philip Bennett, who was taken from us too soon, but provided me with nothing but love and support during the time we had together. And finally, to my mother, Alice Bennett, who has told me every day of my life that I can do anything I set my mind to, for her unconditional love and encouragement. I would never have achieved any of this without her unrelenting support.

5 TABLE OF CONTENTS List of Tables...iv List of Figures...v List of Abbreviations...vi Abstract...x Chapter 1: Cancer Metabolism Chapter Synopsis Cellular Energy Metabolism Carbohydrate and Glucose Metabolism Fat Metabolism and the Ketone Bodies Cancer Metabolism and the Warburg Effect Metastasis Causes and Consequences of Cancer Metabolism Mitochondrial Dysfunction Altered ROS Production and Redox Status Tumor Hypoxia Generation of Biosynthetic Precursors Lactate Production and Acidification of the Tumor Microenvironment Oncogene and Tumor Suppressor Gene Interplay Metastasis and Cancer Metabolism Closing Remarks References for Chapter Chapter 2: Targeting Cancer Metabolism with Ketosis and Hyperbaric Oxygen Chapter Synopsis Targeting Cancer Metabolism with the Ketogenic Diet Literature Review Anti-Cancer Effects of the Ketogenic Diet Decreased Glucose and Insulin Signaling Reduced Flux through the Pentose Phosphate Pathway Influence on ROS Production and Redox Status...53 i

6 Inhibition of Inflammation Potential Concerns or Side Effects Variability in the Effects of the Ketogenic Diet Cancer Cachexia and Weight Loss Effects on Cardiovascular Health Targeting Cancer Metabolism with Ketone Supplementation Exogenous Ketone Supplementation ,3-Butanediol Ketone Esters Anti-Cancer Effects of Ketone Supplementation Inhibition of Glycolysis Influence on ROS Production and Redox Status Histone Deacetylase Inhibitor Activity Enhancement of Mitochondrial Health and Function Inhibition of Lactate Export Potential Concerns or Side Effects Ketoacidosis and Effect on Blood ph Targeting Cancer Metabolism with Hyperbaric Oxygen Therapy Literature Review Anti-Cancer Effects of Hyperbaric Oxygen as a Cancer Therapy Inhibition of Angiogenesis Inhibition of HIF-1 Signaling Enhanced ROS Production Induction of Mesenchymal-to-Epithelial Transition Potential Concerns or Side Effects Stimulating Angiogenesis Soft Tumor Radionecrosis The VM-M3 Model of Metastatic Cancer Central Hypothesis and Project Aims Closing Remarks References for Chapter Chapter 3 The Ketogenic Diet and Hyperbaric Oxygen Inhibit VM-M3 Cancer in Vitro and in Vivo Chapter Synopsis The Ketogenic Diet and Hyperbaric Oxygen Inhibit VM-M3 Cancer in Vitro and in Vivo References for Chapter Chapter 4 Ketone Supplementation Inhibits VM-M3 Cancer in Vitro and in Vivo Chapter Synopsis Ketone Supplementation Inhibits VM-M3 Cancer in Vitro and in Vivo Independently of Glucose Concentration ii

7 4.3 Effect of Ketone-Supplemented Ketogenic Diets on VM-M3 Cancer Effect of Chronic Oral Administration of Ketone Supplements in Rats References for Chapter Chapter 5 Combination Ketosis and HBOT Inhibits VM-M3 Cancer in Vitro and in Vivo Chapter Synopsis Combination Metabolic Therapy Inhibits VM-M3 Cancer in Vitro and in Vivo References for Chapter Chapter 6: Discussion Implications for Cancer Therapy Chapter Synopsis Implications for Cancer Therapy References for Chapter Appendices Appendix A: Materials and Methods Appendix B: Copyright Permissions Appendix C: Published Manuscripts About the Author...End Page iii

8 LIST OF TABLES Table 4.1 Global metabolomics profiling of serum from rats following chronic administration of KE Table A.1 Macronutrient profile and caloric density of diets and ketone supplements iv

9 LIST OF FIGURES Figure 3.1 The KD and HBOT increases mean survival time in mice with systemic metastatic cancer...97 Figure 3.2 Tumor bioluminescence in mice...98 Figure 3.3 Effect of KD, HBOT, and KD+HBOT treatment on metastatic spread in VM-M3 mice...99 Figure 3.4 Blood glucose and β-hydroxybutyrate levels in animals Figure 3.5 Animal weight Figure 3.6 Glucose and weight change are correlated to survival Figure 3.7 Effect of KD+HBOT treatment on metastatic liver tumor size and vascularization Figure 3.8 HBOT inhibits HIF-1α expression and enhances pakt expression in VM- M3 cells Figure 4.1 βhb decreases VM-M3 cell proliferation and viability in vitro Figure 4.2 βhb inhibits HIF-1α and pakt expression in VM-M3 cells Figure 4.3 Effect of supplemental ketones on tumor bioluminescence v

10 Figure 4.4 Supplemental ketones extend survival time in mice with systemic metastatic cancer Figure 4.5 Effect of ketone supplementation on blood glucose, blood βhb, urine AcAc, and body weight in mice Figure 4.6 Decreased blood glucose and weight loss correlated with longer survival Figure 4.7 Blood glucose, βhb, and body weight of KD+BD and KD+KE treated VM-M3 mice prior to significant disease onset Figure 4.8 Effect of KD+BD and KD+KE treatment on tumor growth in VM-M3 mice Figure 4.9 Effect of KD+BD and KD+KE treatment on metastatic spread in VM-M3 mice by in vivo bioluminescent imaging Figure 4.10 Effect of KD+KE treatment on metastatic spread in VM-M3 mice by ex vivo bioluminescent imaging Figure 4.11 Effect of KD+KE treatment on metastatic liver tumor size and vascularization Figure 4.12 Ketone supplemented ketogenic diets extend survival time in VM-M3 mice with metastatic cancer Figure 4.13 Effect of chronic ketone administration on blood glucose in rats Figure 4.14 Effect of chronic ketone administration on blood βhb in rats Figure 4.15 Correlation between blood glucose and blood βhb following oral ketone supplement administration vi

11 Figure 4.16 Effect of chronic ketone administration on blood triglycerides, total cholesterol, HDL, and LDL in rats Figure 5.1 Combination metabolic therapy decreases VM-M3 cell proliferation in vitro Figure 5.2 Combination metabolic therapy decreases VM-M3 cell viability in vitro Figure 5.3 Metabolic therapy increases ROS production and decreases HIF-1α expression in VM-M3 cells Figure 5.4 Blood glucose, βhb, and body weight of VM-M3 survival study mice prior to significant disease onset Figure 5.5 Combination metabolic therapy slows tumor growth and inhibits metastatic spread Figure 5.6 Effect of KD+KE+HBOT treatment on metastatic spread by ex vivo bioluminescence Figure 5.7 Effect of KD+KE+HBOT treatment on metastatic liver tumor size and vascularization Figure 5.8 Combination metabolic therapy extends survival time in mice with metastatic cancer vii

12 LIST OF ABBREVIATIONS AcAc... Acetoacetate ALS... Amyotrophic lateral sclerosis ATA... Atmosphere absolute ADP... Adenosine diphosphate ATP... Adenosine triphosphate βhb... β-hydroxybutyrate CSC... Cancer stem cell CL... Cardiolipin CXCL-1... Chemokine ligand-1 CXCL-2... Chemokine ligand-2 CXCL-3... Chemokine ligand-3 ECM... Extracellular matrix EGF... Epidermal growth factor EGFR... Epidermal growth factor receptor EMMPRIN... Extracellular matrix metalloproteinase inducer EMT... Epithelial-to-mesenchymal transition ETC... Electron transport chain F6P... Fructose-6-phosphate FADD...Fas-associated death domain FADH2... Flavin adenine dinucleotide Fas-L... Fas ligand FDG-PET... Fluorodeoxyglucose positron emission tomography FGF... Fibroblast growth factor vi

13 FH... Fumarate hydratase G6P...Glucose-6-phosphate G6PD... Glucose-6-phosphate dehydrogenase GAPDH... Glyceraldehyde 3-phosphate dehydrogenase GC-MS... Gas chromatography-mass spectrometry GLUT... Glucose transporter GM-CSF... Granulocyte macrophage colony-stimulating factor GOF... Gain of function GPCR... G protein coupled receptor GPx... Glutathione peroxidase GSH...Reduced glutathione GSSG... Oxidized glutathione GTP... Guanosine triphosphate H2O2...Hydrogen peroxide HBO... Hyperbaric oxygen HBOT... Hyperbaric oxygen therapy HIF-1... Hypoxia inducible factor-1 HK2... Hexokinase 2 HRE... Hypoxia responsive element ICAM-1... Intracellular adhesion molecule-1 IDH... Isocitrate dehydrogenase IFN-γ... Interferon-gamma IGF-1... Insulin-like growth factor-1 IL-1β... Interleukin-1 beta IL-2... Interleukin-2 IL-4... Interleukin-4 IL-6... Interleukin-6 IL-7... Interleukin-7 IL-8... Interleukin-8 vii

14 IL-9... Interleukin-9 IL Interleukin-10 IL Interleukin-12 IL Interleukin-13 IL Interleukin-15 IR... Insulin receptor KE... 1,3-Butanediol diacetoacetate ketone ester Km... Michaelis-Menten constant LC-MS/MS... Liquid-chromatography-tandem mass spectometry LDH... Lactate dehydrogenase LDHA... Lactate dehydrogenase A LOF... Loss of function LP... Lysophospholipid MCP-1... Monocyte chemotactic protein-1 MIP-3... Macrophage inflammatory protein-3 MCT... Medium chain triglyceride MCT... Monocarboxylate transporter MMP... Matrix metalloproteinase mtdna... Mitochondrial DNA NADH...Nicotinamide adenine dinucleotide NADPH... Nicotinamide adenine dinucleotide phosphate NCCN... National Comprehensive Cancer Network NO... Nitric oxide O Superoxide anion OH -... Hydroxyl radical ONOO -... Peroxynitrite OXPHOS... Oxidative phosphorylation PAI-1... Plasminogen activator inhibitor-1 PDGF...Platelet-derived growth factor PHD... Prolyl hydroxylase PI3K... Phosphoinositide-3 kinase viii

15 PKB... Protein kinase B po2... Partial pressure of oxygen PPP... Pentose phosphate pathway R5P... Ribose 5-phosphate RANTES... Regulated on activation, normal T cell expressed and secreted ROS... Reactive oxygen species RTK... Receptor tyrosine kinase SDH... Succinate dehydrogenase SMAD... Sma- and mad-related protein SOD... Superoxide dismutase SLP... Substrate level phosphorylation TGF-β... Tumor growth factor-beta TKTL... Transketolase-like 1 TNF-α... Tumor necrosis factor-α UCP... Uncoupling protein VEGF... Vascular endothelial growth factor VHL... von Hippel Lindau ix

16 ABSTRACT Cancer cells exhibit an abnormal metabolic phenotype characterized by glycolysis and lactate fermentation in the presence of oxygen, a phenomenon known as the Warburg effect. This dysregulated metabolism plays an important role in every aspect of cancer progression, from tumorigenesis to invasion and metastasis. The Warburg effect is a common phenotype shared by most, if not all, cancer types. It is especially prominent in metastatic tumors, which are notoriously resistant to treatment and responsible for the majority of cancer-related deaths. Thus, metabolic therapies which target the Warburg effect could offer novel therapeutic options for most cancer patients, including those with aggressive or late-stage cancers. The ketogenic diet is a high fat, low carbohydrate diet that induces a physiological state of nutritional ketosis decreased blood glucose and elevated blood ketones. It has been investigated as a cancer therapy for its potential to exploit the Warburg effect by restricting glucose availability to glycolysis-dependent tumors, and has been reported to slow cancer progression in some animal models as well as in anecdotal reports and small clinical studies in humans. Interestingly, there is some evidence that the elevation in blood ketones induced by the ketogenic diet contributes to its anti-cancer effects, suggesting that ketone supplementation could possibly inhibit cancer progression on its own. Rapid growth outstrips a tumor s ability to adequately perfuse its tissue, creating regions of tumor hypoxia which exacerbate the Warburg effect and promote a malignant phenotype. x

17 Hyperbaric oxygen therapy is the administration of 100% oxygen at elevated barometric pressure. It supersaturates the blood with oxygen, increasing its diffusion distance into the tissues, and can therefore be used to increase intratumoral po2 and reverse tumor hypoxia. Here we present evidence that the ketogenic diet, ketone supplementation, and hyperbaric oxygen therapy work individually and in combination to slow progression and extend survival in the VM-M3 model of metastatic cancer. This study strongly suggests that these cost effective, non-toxic metabolic therapies should be further evaluated in animal and human studies to determine their potential clinical use. xi

18 CHAPTER 1: CANCER METABOLISM 1.1 Chapter Synopsis Here I provide a brief review of cellular energy metabolism, including the mechanisms of ATP production through the anaerobic and aerobic pathways, with a special emphasis on glucose and ketone body metabolism. The prevalence of these pathways are altered substantially in cancer cells, which exhibit a very unique metabolic phenotype characterized by lactate fermentation in the presence of oxygen. This phenomenon is known as the Warburg effect and the dysregulated metabolic pathways which confer this phenotype play critical roles in every aspect of cancer progression, from tumorigenesis to metastasis. A good understanding of the multifaceted and often underappreciated features of cancer metabolism will be critical for interpreting the rationale behind our proposed therapies and the data presented in the subsequent chapters of this dissertation. Thus, the majority of this chapter will be devoted to an in-depth description of multiple causes and consequences of cancer metabolism, including mitochondrial dysfunction, altered reactive oxygen species (ROS) production and redox status, tumor hypoxia, generation of biosynthetic precursors, lactate production and the acidification of the tumor microenvironment, and oncogene and tumor suppressor gene interplay. This chapter will also address the importance of cancer metabolism in the acquisition of a malignant phenotype, the most life-threatening and difficult to treat aspect of human cancer. Since the Warburg effect is a shared feature of most, if not all cancers, and is especially prominent in metastatic tumors, 1

19 therapies which target this phenotype could be an effective treatment for many cancer patients, including those with aggressive or late-stage cancers. 1.2 Cellular Energy Metabolism Energy metabolism includes the complex set of biochemical pathways which convert the food we eat into a usable form of energy for the cell, namely adenosine triphosphate (ATP). The energy released from the hydrolysis of the terminal phosphate bonds of ATP (7.3 kcal) is used to power nearly all of the chemical reactions in the body. It also provides a source of phosphate for protein phosphorylation, one of the most widely used methods of posttranslational modification and regulation of protein function. ATP can be made by oxidizing several types of metabolic fuels, with carbohydrates and fats being some of the most important. The intermediate metabolites formed by the partial oxidation of the major macronutrients enter the metabolic pathways at various stages and their fates are dependent upon the cellular and systemic energy status, metabolic requirements of the particular cell and tissue type, and presence of oxygen, among other factors. Biosynthesis of ATP occurs with the phosphorylation of adenosine diphosphate (ADP) by one of two methods: substrate level phosphorylation (SLP), which does not require oxygen, and oxidative phosphorylation (OXPHOS), which does. SLP occurs in two steps of glycolysis, and is also used to produce guanosine triphosphate (GTP) in the Kreb s cycle, which can be converted to ATP. OXPHOS involves the oxidation of the NADH and FADH2 hydrogen carriers by the components of the electron transport chain (ETC) on the inner mitochondrial membrane. Electrons are passed between the complexes of the ETC in a series of oxidation-reduction 2

20 reactions. The final electron acceptor of the ETC is molecular oxygen, and its reduction produces H2O. During electron transfer, H + protons are pumped into the inner membrane space. Sequestering H + across the inner membrane creates an electrochemical gradient called the proton motive force which provides the potential energy used to power ATP synthesis Carbohydrate and Glucose Metabolism In a fed state, when eating a standard Western diet, glucose is the primary metabolic fuel for the cell. Dietary carbohydrates are digested to glucose, a six carbon monosaccharide which is transported into the cell by a family of membrane proteins called the glucose transporters (GLUT). Glucose is converted to two pyruvate molecules through a series of nine enzymatic reactions collectively called glycolysis. Two reactions of glycolysis, those catalyzed by phosphoglycerate kinase and pyruvate kinase, produce ATP by SLP. The fate of pyruvate oxidation primarily depends on the cellular oxygen status. When oxygen availability is limited, such as in the exercising muscle, pyruvate is converted to lactate by lactate dehydrogenase. This pathway is called anaerobic respiration, or fermentation. It produces another ATP by SLP and replenishes the hydrogen carrier NAD + which is reduced during glycolysis. Under normal oxygen conditions, the cell will preferentially utilize aerobic respiration in the mitochondria to oxidize pyruvate. Pyruvate is converted to acetyl CoA via the pyruvate dehydrogenase complex and shuttled into the mitochondria to participate in the Kreb s cycle. The Kreb s cycle mediates the complete oxidation of the remaining carbons from glucose, producing CO2 and reducing NAD + to NADH+H + and FAD to FADH2. These hydrogen carriers participate in mitochondrial energy metabolism in the aforementioned steps. Aerobic respiration is a much more efficient 3

21 mechanism of energy production than fermentation, producing nearly ten times as much ATP per glucose molecule. Thus, OXPHOS is responsible for approximately 90% of the ATP produced in mammalian cells. When blood glucose levels are low, such as during carbohydrate or calorie restriction, glucose is released from glycogen stores in the muscle and liver. When energy symbols like ATP, NADH, and citrate are high, glucose metabolites are shunted towards fatty acid synthesis to contribute to stored fat reserves Fat Metabolism and the Ketone Bodies Fatty acids from dietary or stored fats undergo β-oxidation to produce acetyl CoA which can combine with oxaloacetate to enter the Kreb s cycle as citrate. The metabolic fate of acetyl CoA from fatty acids is the same as the previously described steps in glucose metabolism. When glucose and insulin levels are low, such as during carbohydrate or calorie restriction or starvation, oxaloacetate in the liver is shipped into the cytosol to participate in gluconeogenesis. Thus, acetyl CoA from fatty acid oxidation does not enter the Kreb s cycle and is shunted towards ketogenesis instead. A series of three enzymatic reactions converts acetyl CoA to acetoacetate (AcAc), one of the three major ketone bodies, or ketones. AcAc can be reduced to produce β-hydroxybutyrate (βhb) or decarboxylated to produce acetone, the two other ketone bodies. Ketones are produced by the liver and released into circulation where they are taken up by extra-hepatic tissues and metabolized for energy. Ketones are transported directly into the mitochondria where they are converted back into acetyl CoA which enters the Kreb s cycle, bypassing glycolysis altogether. From here, the fate of acetyl CoA is the same as in glucose 4

22 metabolism: the Kreb s cycle produces NADH+H + and FADH2 which provide electrons for use in the ETC, establish the proton motive gradient, and power ATP synthesis through OXPHOS. 1.3 Cancer Metabolism and the Warburg Effect In 2000, Hanahan and Weinberg published a landmark review describing the fundamental features of neoplasms which they termed the hallmarks of cancer. The hallmarks included sustaining proliferative signaling, evading growth suppressors, activating invasion and metastasis, enabling replicative immortality, inducing angiogenesis, and resisting cell death [1]. In an updated version released in 2011, they added deregulating cellular energetics as an emerging hallmark [2]. Although described as emerging, the altered metabolic phenotype of cancers has been known for nearly a century, long before most of the other hallmarks were discovered or understood. In the 1950s, the German biochemist Otto Warburg reported that, unlike healthy cells, cancer cells undergo high rates of glucose uptake and lactate fermentation in the presence of oxygen [3]. This phenomenon is known as the Warburg effect, and is often also described as aerobic glycolysis. In the decades since, the Warburg effect has been observed in most, if not all, cancer types [4-6]. Cancer cells exhibit glycolytic rates of up to 200 times greater than that of their normal cellular counterparts [7]. Many cancers also exhibit enhanced glutamine oxidation through the Kreb s cycle; however, it is unclear if this phenotype is as ubiquitous as the Warburg effect. The Warburg effect serves as the basis for one of the most important clinical diagnostic tools, the fluorodeoxyglucose positron emission tomography (FDG- PET) scan [8]. Because of their high glycolytic demand, tumors take up radioactively labeled glucose ( 18 F-FDG) at a much higher rate than surrounding normal tissue, allowing visualization 5

23 by positron emission tomography. Attesting to the near universal presence of the Warburg effect, FDG-PET imaging has a 90% or greater specificity and sensitivity for most cancers [9, 10]. 18 F-FDG avidity is exceptionally high in metastatic cancers, making FDG-PET imaging very useful for identifying metastatic lesions [11], and supporting the observation that the Warburg effect correlates strongly with the aggressive and invasive potential of the tumor as well as cancer progression [12, 13] Metastasis Metastasis is the spreading of a cancer from the site of origin to another part of the body. Metastasis is the major cause of morbidity and mortality in cancer patients, responsible for over 90% of cancer-related deaths [14]. Thus, metastasis is a phenotype of cancer that deserves special attention and should be considered within the context of any potential therapeutic strategies. Most cancers metastasize through the blood or lymphatic vasculature, although some tumors shed malignant cells directly into body cavities, a process called transcoelomic metastasis. The specific mechanisms of intravascular metastasis are not fully understood; however, the general steps include degradation and invasion of the basement membrane, intravasation into the vasculature, dissemination and survival in the circulation, extravasation at a distal tissue, and the formation of metastatic lesions there. Epithelial-to-mesenchymal transition (EMT) is a known embryologic program involving a series of phenotypic and molecular alterations in which epithelial cells acquire mesenchymal characteristics. It has been suggested that during tumor development, cancer cells re-activate this latent program to transition to more aggressive phenotype. It is currently the most 6

24 commonly hypothesized mechanism by which primary tumor cells initiate invasion and metastasis [15]. However, some have suggested that EMT in cancer may be a consequence of the in vitro environment, and the degree to which it occurs in vivo remains largely unknown [16]. Loss of epithelial markers such as E-cadherin, desmoplakin, and plakoglobin, accumulation of nuclear β-catenin, and an upregulation of mesenchymal markers including fibronectin and vimentin are often seen in malignant cancer cells in vitro [17-21]. Transcription factors which mediate the changes in epithelial and mesenchymal marker expression include SNAIL, SLUG, TWIST, and ZEB [22-26]. EMT is thought to occur at the invasive front of the tumor under influence from signals released by stromal cells in the tumor microenvironment [27]. Indeed, SNAIL has been shown to be up-regulated preferentially at the tumor-stromal border in larynx and colon carcinomas, and its expression was inversely related to E-cadherin expression [28]. By adopting a mesenchymal phenotype, cancer cells acquire the ability to degrade and invade the basement membrane, and to intravasate into the blood and lymph circulation. It is hypothesized that once the cells arrive in distal tissues, they revert to an epithelial phenotype through a reverse EMT process termed mesenchymal-to-epithelial (MET) transition in order to establish metastatic tumors [29, 30]. Cancers appear to follow a general pattern of metastatic colonization, with certain cancer types preferentially metastasizing to specific target organs. Homing refers to the migration of a cell to a specific tissue or organ and can be used to describe the behavior of metastatic cells. Proposed explanations for metastatic cell homing include the targeted metastasis of tumor cells which express the adhesion molecules for endothelial cells preferentially expressed on the endothelium of specific organs [31]. Similarly, others have suggested that tumors exhibit a 7

25 preference of metastasizing to tissues rich in growth factors or chemokines for which the cancer cells possesses receptors [31]. A less widely known hypothesis regarding the origin of metastatic cancer involves the transformation of or fusion with immune cells, most notably macrophages, in the tumor microenvironment [32-35]. Macrophages naturally perform the activities of metastatic cells by surviving in circulation, and moving into and out of tissues and the vasculature. Interestingly, many human cancers also exhibit many molecular and behavioral characteristics that are otherwise considered specific to macrophages or other immune cells, including expression of certain antigens as well as phagocytic and fusogenic capabilities [32, 33]. Macrophages are naturally fusogenic and will fuse with other macrophages to produce giant multinucleate cells during the immune response. Interestingly, tumor cell-macrophage fusion has been observed both in vitro and in many animal and human tumors in vivo [32, 36-38]. This observation has led to the hypothesis that these fusion events could produce cells which retain the proliferative, neoplastic phenotype of the tumor cell while acquiring the behavioral characteristics of macrophages, thus allowing them to travel through the body, evade the immune system, and colonize distal tissues [35, 36, 38-40]. Indeed, induced fusion of melanoma cells with macrophages in vitro created hybrid cells with enhanced metastatic potential when grown in vivo [40]. Furthermore, as macrophages are mesenchymally-derived, this acquisition could contribute in part to the EMT-like changes observed in many metastatic cells. Compared to the degree of inquiry put forth towards the role of EMT in metastasis, only a very few studies have examined the specific role of macrophages in the origin of cancer metastasis; however, the existing data strongly indicates a need for further investigation into this novel idea. 8

26 Recently, the discovery of cancer stem cells (CSC), a subpopulation of tumor cells with self-renewal and pluripotent capabilities has led to the hypothesis that metastatic lesions may arise specifically from a pool of CSC within the primary tumor [41]. There is now substantial evidence to support the existence of CSC in human cancers and to suggest that these cells are specifically responsible for tumorigenesis, initiation of metastasis, clinical resistance to therapies, and recurrence of tumors following periods of remission. There has been little success in identifying genetic markers which are consistently expressed by CSC; however, the glycolytic phenotype of the Warburg effect is a fundamental feature of these cells [42]. This is not surprising since a glycolytic phenotype is present in normal stem cells of healthy tissues. The recent development of human induced pluripotent stem cells (ipscs), which earned Gurdon and Yamanaka the 2012 Nobel Prize in Medicine, has provided an incredible tool for understanding pluripotency and cellular differentiation, or dedifferentiation, with far-reaching implications for investigation into the CSC population. Indeed, ipscs share many features with CSC. The four transcription factors used to induce pluripotency in Gurdon and Yamanaka s method Oct4, Sox2, Klf4, and c-myc were all previously known for their oncogenic activities [42]. Importantly, studies on ipscs have demonstrated that the reprogramming of somatic cells to pluripotent stem cells requires acquisition of a glycolytic phenotype, while inhibiting glycolysis enzymes prevents this reprogramming event [42-49]. It is possible that the reprogramming events in both ipscs and CSCs are affected by the Crabtree effect (inhibition of respiration by elevated glucose concentrations) which is often a confounding factor in the culture environment [50]; however, it does not appear that this possibility has been adequately examined to date [51, 52]. 9

27 Taken altogether, these data strongly suggest that the Warburg effect is a critical component of tumorigenesis and metastasis and targeting the glycolytic phenotype of cancer cells could provide significant therapeutic potential. Indeed, many aspects of EMT, invasion, and metastasis are influenced by or mediated by cancer metabolism. A discussion of these mechanisms will be included in the following sections Causes and Consequences of Cancer Metabolism As described, fermentation is an inefficient mechanism of energy production compared to OXPHOS. Thus, it is not immediately clear why rapidly proliferating tumors with high metabolic demands would utilize this method. This paradox has prompted researchers to investigate and hypothesize multiple causes and consequences of the Warburg effect which may underlie this unique cancer metabolism. The Warburg effect is a consistent and well-established metabolic phenotype of cancer cells; however, alterations in cancer energy metabolism are complex and require insight far beyond the balance between fermentation and OXPHOS. Some important aspects which should be discussed include mitochondrial dysfunction, altered ROS production and redox status, tumor hypoxia, generation of biosynthetic precursors, lactate production and the acidification of the tumor microenvironment, and oncogene and tumor suppressor gene interplay. 10

28 Mitochondrial Dysfunction The role of the mitochondria in carcinogenesis and tumor progression is multifaceted and remains incompletely understood. The degree to which mitochondria are functional, or dysfunctional, in cancers is a hotly debated topic [52-54]. Unlike in cancer, the majority of ATP energy produced by healthy cells is derived from OXPHOS in the mitochondria. Warburg originally hypothesized that impaired mitochondrial OXPHOS forces cancer cells to acquire their fermentative phenotype (Warburg effect) in order to preserve energy production and maintain viability [3]. He further proposed that this metabolic damage was the instigating cause of tumorigenesis, a proposition known as the Warburg hypothesis [55]. In the decades since Warburg s work and hypotheses, mitochondrial abnormalities have been reported in most if not all tumors examined [53], although its specific role in cancer development remains unclear. A review of 20 studies consistently demonstrated decreased mitochondrial numbers in tumor samples compared to healthy tissue of the same origin [56]. Other studies reported that decreased mitochondrial DNA (mtdna) copy number is associated with higher histological grade, propensity for metastasis, and worse clinical prognosis and overall survival in patients with breast cancer and osteosarcoma [57, 58]. Mitochondria have an important morphological structure which facilitates their function. Electron transport occurs along the folds of the inner mitochondrial membrane called cristae, maximizing surface area for optimal ATP production. Abnormalities in mitochondrial morphology are indicative of mitochondrial dysfunction and correlated with respiratory insufficiency [59]. The morphology of tumor mitochondria vary significantly from that of healthy cells. Many studies have shown that tumor mitochondria often exhibit partial or total cristolysis. Ultrastructural analysis of human brain tumors by electron microscopy found abnormal mitochondrial morphology in all samples studied [60]. 11

29 Mitochondrial swelling and partial or total cristolysis of the inner membrane was the most consistent phenotype observed. In a similar study of almost 800 breast tumor biopsies, approximately 60% of tumor samples examined lacked any identifiable mitochondria [61]. Of the 40% of tumors in which mitochondria were present, half contained only very few mitochondria which were morphologically abnormal swollen, vacuolated, and exhibiting cristolysis. Interestingly, the degree of mitochondrial dysfunction correlated with a more anaplastic and aggressive histological phenotype. The lipid composition of the inner mitochondrial membrane also differs widely between cancer cells and their normal counterparts, with abnormalities in cholesterol and specific phospholipid content [62, 63]. Cardiolipin (CL) is a mitochondrial specific phospholipid that regulates electron transport, OXPHOS, and is essential for mitochondrial coupled respiration [64-71]. Seyfried and colleagues demonstrated that CL content is significantly lower in brain tumor mitochondria compared to normal brain tissue in mice [72]. Similar results were found in rhabdomyosarcoma cells, and in both studies, CL abnormalities were linked to decreased complex I activity [72, 73]. Reduced expression of or mutations in the genes encoding the ETC complexes have been reported in several different cancers as well [50, 53, 74-77]. Furthermore, decreased expression of the β-subunit of ATP synthase, the mitochondrial enzyme which couples electron transport to ATP synthesis via OXPHOS, has been reported in liver, kidney, colon, breast, gastric, oesophageal, and lung carcinomas [78, 79]. Mitochondrial uncoupling refers to the dissipation of the proton motive gradient across the inner mitochondrial membrane and is characterized by an uncoupling of electron transport from ATP synthesis by OXPHOS. Uncoupling can be induced by uncoupling proteins (UCPs) or can be a consequence of inner membrane dysfunction caused by abnormal lipid composition, 12

30 most notably CL. Several studies have demonstrated that mitochondrial uncoupling occurs in tumors of varied tissue types. UCP expression is up-regulated in some tumors [80, 81]. One study showed that H + back-decay the leaking of H + protons into the mitochondrial matrix from the inner membrane space was eightfold greater in Ehrlich ascites tumor mitochondria compared to normal mitochondria [82]. Brown adipose tissue contains naturally uncoupled mitochondria which use electron transport to generate heat for thermoregulation rather than produce ATP by OXPHOS. Therefore, heat production is considered a hallmark of mitochondrial uncoupling. Many human tumors generate heat, leading to the investigation of thermography, or heat mapping, as a potential diagnostic tool [83]. In a study in human patients with melanoma, thermography differentiated malignant melanoma lesions from benign lesions with significant sensitivity and selectivity [84]. Numerous other mitochondrial abnormalities are reported in cancers across tissue type, including mtdna mutations [85, 86], abnormal fissionfusion [60], and altered mitochondrial membrane potential [87-89]. Further support of mitochondrial dysfunction in cancer is illuminated by studies in which transfer of healthy mitochondria into cancer cells suppress the neoplastic phenotype [90-93], leading some to suggest that healthy mitochondria function as a tumor suppressor [52]. It is important to note that aside from the metabolic responsibilities of the mitochondria, they are also critically involved in calcium homeostasis, control of cell death, and redox status three phenomenon which are also drastically altered in cancers [94]. Taken together, these data strongly suggest that decreased capability and/or contribution of mitochondrial respiration and function is a fundamental feature of cancer. Despite such an overwhelming body of evidence, many researchers adhere to the notion that mitochondrial respiration is not impaired and the Warburg effect is simply an adaptive phenotype [10]. This 13

31 hypothesis is based largely on studies which demonstrate that cancer cells continue to consume oxygen and produce CO2. It is important to note that although respiring mitochondria will consume O2 and produce CO2, it is not necessarily true that oxygen consumption is coupled to ATP production. This is especially true for uncoupled mitochondria. Indeed, studies have shown that oxygen consumption is greater in cells with greater tumorigenic potential, but these cells rely less heavily on mitochondrial respiration compared to those with less tumorigenic potential [95]. There is no sophisticated method to determine exactly what proportion of cellular ATP is derived from SLP in glycolysis and the Kreb s cycle compared to functional OXPHOS in the mitochondria. However, it is unclear and unlikely that tumors displaying the wide variety of mitochondrial abnormalities previously described could maintain normal respiration. As such, many scientists agree with Warburg s original hypothesis: that the Warburg effect and associated metabolic phenotype of cancer is primarily caused by mitochondrial dysfunction and respiratory insufficiency [52, 96] Altered ROS Production and Redox Status As electrons are transferred between the complexes of the mitochondrial electron transport chain (ETC), some are captured by molecular oxygen to form the superoxide anion (O2 - ). O2 - is dismutated to hydrogen peroxide (H2O2) by the superoxide dismutase (SOD) enzymes. H2O2 is less damaging than O2 - and is freely permeable through cellular membranes, making it a useful cellular signaling molecule. In the presence of iron, H2O2 reacts to form the hydroxyl radical ( OH) via the Fenton reaction. O2 - can react with another free radical, nitric oxide (NO), to form peroxynitrite (ONOO - ). OH and ONOO - are highly reactive. They induce oxidative 14

32 damage onto DNA, proteins, and lipids and are responsible for much of the toxicity associated with ROS. Although once thought of only as toxic byproducts of metabolism, it is now understood that ROS serve as important signaling molecules in healthy cells. Low levels of ROS are produced during normal mitochondrial metabolism while damaging levels of potent oxidizers are kept in check by endogenous antioxidant systems like SOD, catalase, and glutathione peroxidase (GPx) [97, 98]. It is thought that ROS production increases when electron transport is inhibited in the ETC [99, 100]. Mutations in the nuclear and/or mitochondrial genes encoding the ETC complexes increase ROS production in cancers [99]. It is well established that basal elevation of ROS is a consistent phenotype in cancers across tissue types [101], likely due to widespread mitochondrial damage [102]. Chronic inflammation from sustained hyperglycemia, infection, or exposure to chemicals like tobacco, is also a major source of ROS production in tumors [101]. Elevated ROS confer a growth advantage to the tumor in many ways and play a significant role in tumorigenesis and progression [103]. Growth factor signaling often works through receptor tyrosine kinase (RTK) cascades to drive cell proliferation. Several of these RTKs act through ROS-dependent mechanisms. Epidermal growth factor receptor (EGFR) and platelet-derived growth factor (PDGF) signaling work in part through H2O2 signaling mechanisms [104, 105]. H2O2 oxidizes PTEN to prevent inactivation of RTK signaling through Akt. ROS-induced stabilization of HIF-1 enhances the transcriptional activation of numerous glycolytic, proliferative, and pro-survival genes [106]. Furthermore, mtdna is especially vulnerable to oxidative damage due to its close proximity to ETC-generated ROS [107]. Like nuclear mutations, mtdna mutations are heavily implicated in malignant transformation and cancer progression [ ]; however, not all tumors contain pathological mtdna mutations [111]. 15

33 The nuclear genome is also a major target of ROS, creating widespread mutations and DNA strand breaks. This enhanced mutability further contributes to the collection of oncogenes and tumor suppressor gene mutations in tumor cells which undergo clonal expansion to produce a more malignant phenotype [112]. The cancer-promoting effects of ROS have prompted efforts to develop therapies which inhibit ROS production by tumors [113]. This chronic elevation in ROS production is kept at sub-lethal levels by an up-regulation of endogenous antioxidant systems [97, 98], allowing cancers to survive and thrive in a redox state which would be toxic to healthy cells. There still exists, however, a threshold point above which ROS will irreversibly damage and induce apoptosis in cancer cells [114]. While the cancer-promoting effects of ROS have incited the proposal and testing of anti-oxidant therapies for cancer, recognition of cancer susceptibility to further oxidative stress have spurred development of pro-oxidant therapies with the intention of pushing cells past their oxidative breaking point to incite cell death [113]. The antioxidant ascorbic acid (vitamin C) is being investigated in clinical trials as a potential cancer therapy [115], but it may actually work as a pro-oxidant at very high doses [116]. All current non-surgical standard therapies, including radiation and chemotherapy, work in part by enhancing ROS production [113]. This paradoxical phenomenon has led to the common understanding that ROS is truly a double-edged sword for cancer, which can be exploited by both the tumor, researcher, and patient in multiple ways to either promote or inhibit malignant progression [99]. 16

34 Tumor Hypoxia Hypoxia in solid tumors is a well-documented phenomenon [117]. Normal tissue oxygen tension is highly tissue-specific, but typically ranges between mmhg [118]. Intratumoral oxygen concentrations vary widely, ranging on average from 2.5 to 30 mmhg, with remarkable heterogeneity even within a single tumor [118]. In order to grow larger than approximately 1mm 3, tumors must stimulate angiogenesis to supply blood and nutrients to the growing tumor [119]. Under the influence of chaotic signaling cascades induced by oncogene activation, these new blood vessels are often malformed, immature, leaky, and unable to adequately perfuse the entire tumor [120]. Regions of the tumor which are not adequately vascularized become either transiently or chronically hypoxic [117]. Hypoxia is present even in small tumors, and preclinical studies have demonstrated that tumors that grow to 4 to 10 mm in diameter exhibit very large hypoxic regions [121, 122]. Oxygen tensions in the center of large tumors often approach 0 mmhg, creating areas of central necrosis [123]. Tumor hypoxia dramatically affects malignant progression and therapeutic response. It has been correlated to poor clinical prognosis in a variety of human cancers, including head and neck, uterine, cervical, and soft tissue sarcomas [ ]. Radiation and many chemotherapies work by causing tumors to over-produce ROS which kill the cell by inflicting widespread lipid, protein, and DNA oxidative damage and inducing apoptosis and mitochondrial damage. Hypoxia stimulates ROS production largely for signaling purposes [127]. However, since oxygen is the substrate for ROS generation, hypoxia effectively prevents large quantities of ROS from being produced in response to therapeutic insult [128]. Indeed, the efficacy of radiation therapy is directly proportional to tissue po2 [128]. The insufficient blood supply which induces hypoxia also prevents drug delivery to avascular regions within the tumor, further contributing to 17

35 chemotherapy resistance [129]. Rather than succumbing to treatment, hypoxic cells survive by entering a non-proliferative state [130]. After removal of therapeutic pressure, these hypoxic cells undergo clonal expansion to repopulate recurrent tumors which often have a highly malignant phenotype and are resistant to subsequent treatment [ ]. Cancerous tumors adapt to nutrient and oxygen deprivation by three major mechanisms activating angiogenesis, inhibiting apoptosis, and induction of the glycolytic shift which eventually allow them to escape the confines of the primary tumor and metastasize to distal regions in the body [118]. These adaptations are mediated in part through tumor hypoxia and expression of the hypoxia inducible factor-1 (HIF-1) transcription factor. Stability of HIF-1α, the regulatory subunit of HIF-1, is dependent on tissue po2. Under normal oxygen tensions, proline residues on HIF-1α are hydroxylated by prolyl hydroxylases (PHDs) which serve as a major oxygen sensing mechanism in the cell [135]. This hydroxylation event allows HIF-1α to be recognized, ubiquitinated, and targeted for proteasomal degradation by the von Hippel Lindau (vhl) E3 ubiquitin ligase and tumor suppressor. During hypoxia, HIF-1α is not hydroxylated by PHDs nor ubiquitinated by VHL, but rather it accumulates and forms complexes with HIF-1β and other complex subunits to form the functional HIF-1 transcription factor. HIF-1 translocates to the nucleus where it directly regulates the expression of over 60 genes involved in energy metabolism and survival [136, 137]. However, the pleiotropic response to HIF-1 can alter the expression of nearly 500 genes [138]. HIF-1 binds to a conserved sequence in the promoter region of HIF-responsive genes called hypoxia responsive element (HRE). In healthy tissues, HIF-1 signaling serves as a mechanism to promote survival during mild or transient hypoxia, such as in the exercising muscle. Chronic or severe hypoxiainduced HIF-1 signaling will initiate apoptosis through p53 [139], but this pathway is commonly 18

36 inactivated in malignant cells [140]. Rather, the dysregulated vascular environment of hypoxic tumors stimulates HIF-1 activity which confers potent growth and survival benefits without proper inhibitory feedback, promoting unbridled proliferation, invasion, and metastasis [136, 141]. Experimentally, HIF-1α gain of function (GOF) manipulations increase tumor growth rate, angiogenesis, and metastasis while LOF elicits the opposite effects [106]. HIF-1 signaling in cancer is extensive, and its role has been expertly reviewed [106, 142]. Collectively, these studies show that HIF-responsive genes play vital roles in virtually every aspect of cancer biology, including immortalization [143], resistance to apoptosis [126], genomic mutability [144], immune evasion [145], tumor growth and proliferation [146], angiogenesis [147], energy metabolism [136], ph regulation [148], dedifferentiation [149], radio- and chemo-resistance [150], EMT [151], invasion [152], and metastasis [153]. The Angiogenic Switch. During hypoxia, angiogenic factors are secreted by tumor and stromal cells, including endothelial cells, fibroblasts, and infiltrating immune cells. Vascular endothelial growth factor (VEGF) is one of the most potent angiogenic factors and is the most commonly expressed cytokine induced by hypoxia [154]. HIF-1 signaling enhances the expression of VEGF. High blood levels of VEGF is correlated with poor prognosis in pancreatic, prostate, breast, renal, colorectal, and head and neck cancers [ ]. The blood vessels formed by VEGF signaling in tumors are immature and facilitate chaotic blood flow, which in turn cause further hypoxia. This creates a positive feedback system wherein hypoxia can be considered both a cause and consequence of tumor angiogenesis [118]. The expression of many other angiogenic factors is enhanced during the angiogenic switch, including plateletderived growth factor (PDGF), angiogenin, epidermal growth factor (EGF), fibroblast growth factor (FGF), and transforming growth factor-β (TGF-β) [118]. Many of these signaling 19

37 molecules also stimulate growth and proliferation. In this way, the signaling initiated by the angiogenic switch promotes tumor growth while simultaneously stimulating the new blood vessel formation which is required for further tumor growth. Subsequently, the immature blood vessels formed by this cycle exacerbate the hypoxic signaling which ensures further growth factor stimulation. Deregulation of Apoptosis. Cancer cells will increase their metabolic rate to maintain anabolic processes and energy production during hypoxia [118, 162, 163]. Mitochondrial damage exacerbates hypoxia-induced ROS production, which contributes to DNA damage and the inactivation of important tumor suppressor pathways [102]. While normal cells possess functional cell cycle checkpoints and repair mechanisms to prevent cell cycle progression or induce apoptosis in the face of DNA damage, the enhanced mutability of cancer cells eventually selects for cells which have deactivated these protective mechanisms. p53 and Rb are two of the most important regulators of DNA damage response mechanisms and both are commonly mutated or functionally absent in cancers [140, 164]. Chronic or severe hypoxia initiates cell death through HIF-1 stimulation of p53 in healthy tissues, but fails to do so in cancer cells with dysregulated p53. Hypoxia also up-regulates anti-apoptotic Bcl-2 family members like Bcl-2 and Bcl-Xl, while inhibiting translocation of the pro-apoptotic Bax to the mitochondrial membrane, further establishing resistance to apoptosis [130, 165, 166]. Thus, tumor hypoxia promotes the clonal expansion of highly malignant cells that are resistant to cell death. Glycolytic shift. The faulty vasculature in growing tumors induces not only oxygen deprivation, but nutrient deprivation as well. Normally, approximately 90% of cellular ATP is produced by mitochondrial aerobic respiration, an oxygen-dependent process. During hypoxia, activation of HIF-1 enhances the transcription of glucose transporters (GLUT) and glycolytic 20

38 enzymes to maintain energy production via substrate level phosphorylation [136, 141, 167]. Pyruvate from glycolysis is not oxidized in the mitochondria, but is shunted towards lactate fermentation to produce minimal ATP and replenish NAD +. In order to satisfy cellular energy requirements, glycolysis and fermentation rates must increase significantly. The Warburg effect describes how this phenomenon occurs in tumors regardless of tumor oxygenation. When present, however, tumor hypoxia enhances glycolysis, fermentation, and the Warburg effect. Thus, hypoxia stimulates the glycolytic shift which preserves energy production to support cellular function and survival in response to nutrient deprivation. Invasion and Metastasis. Individually, hypoxia and EMT have been considered crucial events in cancer progression and acquisition of a metastatic phenotype. Recent evidence suggests, however, that alterations in tissue po2 and the hypoxic signaling mechanisms mediated by HIF may be crucial triggers and mediators of EMT. Hypoxia significantly enhances the production of TGF-β in myeloid, mesenchymal, and cancer cells [ ]. TGFβ works through both SMAD and non-smad pathways to promote tumor progression and invasion, and mediate EMT. TGF-β-SMAD signaling induce the expression of SNAIL and ZEB. SMAD3/4 complex with SNAIL to inhibit the expression of E-cadherin [172]. SMADs also regulate the activation of β-catenin to induce EMT [ ]. Non-SMAD TGF-β signaling cross-talks with other oncogenic pathways like Ras/MAPK, Wnt/β-catenin, and NFκB to promote acquisition of the mesenchymal phenotype [ ]. The Notch pathway is important for stem-cell maintenance during development and regulation of differentiation in adult tissues. Notch is also influenced by hypoxia [182, 183]. Notch signaling has been shown to mediate hypoxia-induced motility and invasion in cervical, ovarian, colon, and glioma cells [184]. HIF-1 activation also 21

39 mediates EMT by modulating the expression of the SNAIL, SLUG, TWIST, and ZEB transcription factors [151, 185] Generation of Biosynthetic Precursors The pentose phosphate pathway (PPP) is a divergent metabolic pathway from glycolysis which plays an important role in cellular proliferative rate and redox balance. Rather than being isomerized to fructose-6-phosphate (F6P) in glycolysis, glucose-6-phosphate (G6P) is converted to ribose 5-phosphate (R5P) through a series of enzymatic reactions, generating NADPH in the process. R5P and NADPH are important biosynthetic precursors for nucleotide and lipid synthesis, respectively. Thus, PPP activity is significantly enhanced for increased cellular proliferation [186]. One consequence of the increased glycolytic rate associated with the Warburg effect is increased flux through the PPP. In cancer cells, the PPP provides approximately 85% of the pentose phosphates incorporated into newly synthesized DNA [187]. Glucose-6-phosphate dehydrogenase (G6PD), the enzyme responsible for shuttling G6P into the PPP, is up-regulated in many cancers and is now considered to be an oncogene [188]. p53 is an inhibitor of G6PD [189], but its common inactivation in tumors allows dysregulated and excessive PPP flux [190]. The PPP also plays a significant role in maintaining endogenous antioxidant capacity, as NADPH is required for regeneration of reduced glutathione (GSH). GSH prevents oxidative stress by neutralizing damaging free radicals. As described, tumors thrive in a state of elevated basal ROS which promotes their growth and proliferation [99]. Enhanced GSH protects cancer cells against oxidative stress-induced apoptosis. Since radiation and many chemotherapies work 22

40 by overproducing ROS in the tumor, elevated flux through the PPP is widely considered to mediate some degree of clinical treatment resistance [186]. Indeed, G6PD expression is further enhanced following ionizing radiation and confers protection against radiation-induced cell death [191] Lactate Production and the Acidification of the Tumor Microenvironment As described, much of the pyruvate generated by glycolysis in cancer cells is converted to lactate by lactate dehydrogenase (LDH). At physiological ph, lactic acid exists primarily as a dissociated lactate anion and proton. To prevent intracellular acidification and death, lactate is exported from the cell by a family of passive proton-lactate membrane symporters called the monocarboxylate transporters (MCTs). Not surprisingly, MCTs, especially the MCT1 and MCT 4 isoforms, are over-expressed in most cancers to facilitate the overload of lactate production that occurs with the Warburg effect [192, 193]. While the normal concentration of lactate in healthy tissue is approximately 2 mm, the median value of human tumors is 14 mm and can range up to 40mM [ ]. Previously the excess production of lactate by cancers was considered simply to be a byproduct of the Warburg effect, but we now know that lactate mediates a considerable number of effects in the tumor, promoting growth, invasion, and metastasis [12]. Lactate itself is an important signaling molecule, inducing the activity of over 650 genes involved in a wide variety of cellular functions, such as signal transduction, membrane transport, oxidative stress, growth, proliferation, and apoptosis [197]. Lactate is also considered to be a hypoxia mimetic, as it is able to activate HIF-1 in normoxic cancer cells, suggesting multiple 23

41 mechanisms by which lactate promotes tumor progression, invasion, and metastasis [106, 198, 199]. Extracellular acidification promotes angiogenesis by inducing the expression of VEGF and IL-8, further facilitating tumor growth (99, 100). The expression and function of the MCT proton/lactate symporters are largely dependent upon interaction with the extracellular membrane protein CD147 [200]. CD147 is also known as extracellular matrix metalloproteinase inducer (EMMPRIN), a name which reflects its important role in the remodeling of the extracellular matrix [201]. CD147 stimulates production of matrix metalloproteases (MMPs) by the tumor as well as stromal fibroblasts and endothelial cells [202]. These MMPs are activated by the low ph of the tumor microenvironment induced by lactate export [203]. Other modulators of the ECM are also activated by the acidic tumor microenvironment, including urokinase-type plasminogen activator and the cathepsins [ ]. These enzymes degrade the basement membrane of the ECM, providing a pathway for invasion and metastasis. The importance of lactate in tumor progression and analysis is further supported by in vivo studies in which inhibition of the MCT transporters and/or CD147 reduces tumor growth and invasion [202, ]. Human tumors which are commonly associated with metastatic spread have higher lactate levels than non-metastatic tumors [194, 195, ]. Considering this, it is not surprising that elevated lactate is clinically correlated to poor prognosis and a decrease in overall survival in a variety of cancers, including cervical, brain, head and neck, and lung [195, 216, 217, ]. 24

42 Oncogene and Tumor Suppressor Gene Interplay P53 is a well-characterized tumor suppressor that plays important roles in DNA damage and repair, and cell cycle control; however, its role in energy metabolism has only recently been characterized. P53 stimulates aerobic respiration by activating the SCO2 gene which encodes the cytochrome c oxidase assembly protein and is thus required for assembly of the cytochrome c oxidase complex, an important component of the ETC [223]. Therefore, loss of p53, a common feature of many cancers, inhibits mitochondrial respiration [224]. This induces an increased reliance on fermentation for energy production, contributing to the Warburg effect. Akt, also known as Protein kinase B (PKB), is a serine/threonine kinase and oncogene that stimulates proliferation and promotes cell survival by inhibiting apoptosis [225, 226]. Recently, it was shown that Akt enhances glucose uptake by inducing the translocation of the GLUT transporters to the plasma membrane [227]. It also activates hexokinase 2 (HK2), the first glycolytic enzyme which phosphorylates glucose to trap it in the cell, thus contributing to the Warburg effect. Overexpression of the oncogenic transcription factor c-myc is also present in many different cancer types [228]. Myc activation promotes proliferation and assists in the maintenance of the stem-like phenotype [143, 229]. Myc contributes to aerobic glycolysis by binding to and activating nearly all of the glycolytic enzyme genes, including HK2, enolase, and lactate dehydrogenase A (LDHA) [230]. Furthermore, the acquisition of oncogenes and loss of tumor suppressors often activate HIF-1 which, as previously described, contributes to the Warburg effect through numerous mechanisms. Oncogenes which stabilize HIF-1 include Src, Ras, PI3K, Akt, mtor, EGFR, and HER2 [142, ]. Similarly, loss of the PTEN, p53, and VHL tumor suppressors stabilize HIF-1 as well [232, 233, 236]. 25

43 It has recently been discovered that mutations in metabolic enzymes alone, such as the Kreb s cycle enzymes isocitrate dehydrogenase 1 (IDH1), fumarate hydratase (FH), and succinate dehydrogenase (SDH), are able to initiate tumors in vivo [237]. SDH mutations are common in gastric tumors, T-cell leukemia, and head and neck cancers [238, 239], while over 70% of gliomas and glioblastomas contain IDH mutations [240, 241]. It is thought that the loss of some of these metabolic tumor suppressors is also due to their effects on HIF-1. SDH and FH mutations cause an accumulation of succinate and fumarate which are structural analogs of α- ketoglutarate (α-kg). Succinate and fumarate appear to mimic the effects of α-kg by inhibiting PHDs to activate the HIF-1 pathway [242, 243]. Furthermore, mutations in these metabolic enzymes would impair mitochondrial respiration and may have similar cancer promoting and metabolism altering effects as the aforementioned mitochondrial dysfunction. Interestingly, new evidence suggests that some oncogenes elicit their tumorigenic effects by mediating the Warburg effect. V600E BRAF is a major driver mutation in the MAPK pathway of melanoma cells which induces proliferative signaling. Hall and colleagues recently demonstrated that V600E BRAF also works to compensate for OXPHOS dysfunction by increasing glycolytic activity [244]. Although V600E BRAF activation sustains proliferative signaling through its effects on the MAPK pathway, this is not its critical function in melanoma. Rather, oncogene addiction to V600E BRAF appears to be more fundamentally a glycolytic addiction as cellular senescence induced by V600E BRAF inhibition is rescued by overexpression of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [244]. Interestingly, the Warburg effect can also induce the activation of oncogenes. Overexpression of GLUT3 in normal human breast cells activates a number of oncogenic pathways, including MAPK, Akt, EGFR, induces loss of apicobasal polarity, and enhances cell growth [245]. 26

44 1.3.3 Metastasis and Cancer Metabolism The previous sections describing the causes and consequences of cancer metabolism illustrate how the dysregulated metabolic phenotype of cancer plays a critical role in every step of cancer progression, from tumorigenesis to invasion and metastasis. Metastasis is considered the final phase of cancer development, and it is inarguably the most dangerous and least treatable aspect of the neoplastic phenotype. Mitochondrial dysfunction, ROS production, tumor hypoxia, lactate production, and metabolically-mediated oncogene and tumor suppressor gene alterations all aid in the transition to a malignant phenotype. Some researchers even hypothesize that the Warburg effect is primarily an adaptive mechanism to mediate acidification of the tumor microenvironment and allow for a primary tumor s escape from the space limitations of the site of origin [246]. As described, the Warburg effect appears to be a prevalent and universal feature of metastatic tumors. 18 F-FDG uptake and lactate production are both strongly correlated with metastasis and poor clinical outcome while mediators of cancer s metabolic phenotype [12, 13, 246], most notably HIF-1, regulate numerous players in EMT [20, 247]. The sheer prevalence of the Warburg effect in metastatic tumors demonstrate its importance in the malignant process and strongly suggests that therapies which exploit these metabolic pathways could offer new hope for the treatment of aggressive or late-stage cancers. 1.4 Closing Remarks It is clear from a comprehensive review of the literature that cancer metabolism is a critical component of the neoplastic phenotype. Recognition of the importance of these features provides insight into novel therapeutic targets aside from the classically-targeted genetic 27

45 mutations of cancer. The ubiquitous nature of the Warburg effect in cancer, and the unique metabolic differences between healthy and cancer cells, have led to the suggestion that metabolism may provide a long-awaited Achilles heel to target therapeutically [248]. In the following chapter we will discuss three potential therapies which exploit cancer metabolism: the ketogenic diet, ketone supplementation, and hyperbaric oxygen therapy. 1.5 References for Chapter 1 1. Hanahan D, Weinberg RA: The hallmarks of cancer. Cell 2000, 100(1): Hanahan D, Weinberg R: Hallmarks of cancer: the next generation. Cell 2011, 144(47c26d71-ae0f-a869-8b1c-9c7fa4d43ff3): Warburg O: On respiratory impairment in cancer cells. Science 1956, 124(3215): Hsu P, Sabatini D: Cancer cell metabolism: Warburg and beyond. Cell 2008, 134: Bayley J-P, Devilee P: The Warburg effect in Current opinion in oncology 2012, 24: Gillies RJ, Gatenby RA: Adaptive landscapes and emergent phenotypes: why do cancers have high glycolysis? Journal of bioenergetics and biomembranes 2007, 39(3): Pecqueur C, Oliver L, Oizel K, Lalier L, Vallette FM: Targeting metabolism to induce cell death in cancer cells and cancer stem cells. International journal of cell biology 2013, 2013: Wijsman R, Kaanders JH, Oyen WJ, Bussink J: Hypoxia and tumor metabolism in radiation oncology: targets visualized by positron emission tomography. The quarterly journal of nuclear medicine and molecular imaging : official publication of the Italian Association of Nuclear Medicine 2013, 57(3): Czernin J, Phelps ME: Positron emission tomography scanning: current and future applications. Annual review of medicine 2002, 53: Gatenby R, Gillies R: Why do cancers have high aerobic glycolysis? Nat Rev Cancer 2004, 4: Gambhir SS, Czernin J, Schwimmer J, Silverman DH, Coleman RE, Phelps ME: A tabulated summary of the FDG PET literature. J Nucl Med 2001, 42(5 Suppl):1S-93S. 12. Dhup S, Dadhich RK, Porporato PE, Sonveaux P: Multiple biological activities of lactic acid in cancer: influences on tumor growth, angiogenesis and metastasis. Curr Pharm Des 2012, 18(10):

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61 CHAPTER 2: TARGETING CANCER METABOLISM WITH KETOSIS AND HYPERBARIC OXYGEN 2.1 Chapter Synopsis Although the Warburg effect has been recognized for nearly a century, the field of cancer metabolism has received most of its interest and attention over the past decade. The new information gained from these studies has revealed a multitude of novel therapeutic targets which exploit the dysregulated metabolic phenotype of cancer. In this chapter, we provide a review of the literature and scientific rationale for three potential metabolic therapies the ketogenic diet (KD), ketone supplementation, and hyperbaric oxygen therapy (HBOT). The KD is a high fat, low carbohydrate diet that is hypothesized to inhibit cancer progression by restricting glucose availability to glycolysis-dependent tumors. The KD also suppresses insulin signaling and flux through the pentose phosphate pathway, and inhibits ROS production and inflammation all which could create an unfavorable environment for cancer. Exogenous ketone supplementation as a cancer therapy is a novel idea that stems from the observation that the elevation of blood ketones induced by the KD may contribute to its efficacy. It may also hinder cancer by its ability to inhibit glycolysis and ROS production, act as a histone deacetylase inhibitor, enhance mitochondrial health, and inhibit lactate export. HBOT is the delivery of 100% oxygen at elevated barometric pressure. It has mostly been investigated clinically as a radiosensitizer, but has shown some efficacy as a stand-alone treatment in animal models, and 44

62 likely works by inhibiting angiogenesis and HIF-1 signaling, enhancing ROS production, and inducing mesenchymal-to-epithelial transition. We hypothesize that these therapies would work individually and in combination to slow cancer progression and propose to test their effects on the VM-M3 model of metastatic cancer. 2.2 Targeting Cancer Metabolism with the Ketogenic Diet Dietary interventions have been used to prevent and treat diseases for millennia. Even when effective, however, dietary therapies are often overlooked or underappreciated in modern medicine [1]. An overwhelming collection of epidemiological studies now show a strong correlation between obesity, sedentary lifestyles, and the consumption of certain diets with lifetime risk of developing cancer [2-5]. Considering the Warburg effect, it is not surprising that many of these studies indicate sugar consumption and high glycemic index diets are correlated with an increased risk of developing a variety of cancer types [6-14]. An 8 year longitudinal study in middle aged Swedish men and women reported that consumption of sugar or high sugar foods, including soft drinks, sweetened fruit soups, or stewed fruits, was associated with a significantly increased risk of pancreatic cancer [14]. Data from a population-based case-control study revealed a significant correlation between high ratio of sucrose to dietary fiber intake and risk of colon cancer [8]. This increased risk was greatest in patients who were sedentary and/or had a high body mass index (BMI), leading authors to suggest that diets high in simple carbohydrates increase colon cancer risk in part by elevating blood glucose levels [8]. At least two large prospective studies demonstrated a significant correlation between the consumption of diets with a high glycemic index with an increased risk of breast cancer among overweight or 45

63 postmenopausal women [12, 13]. Similar to other cancer types, this risk was increased with limited physical activity or increased BMI [12, 13]. It is hypothesized that the frequent postprandial hyperglycemia and hyperinsulinemia caused by the consumption of high glycemic index diets play a causative role in this increased risk of cancer. Indeed, an increase in glucose uptake by premalignant cells may also be sufficient to drive tumorigenesis in vitro. Bissell et al reported that overexpression of glucose transporter type 3 (GLUT 3) in normal human breast cells altered expression of the extracellular matrix mediator β1-integrin leading to a loss of cell polarity, and activated EGFR, MEK, and AKT oncogene signaling pathways leading to increased growth and proliferation [6]. It is not surprising, perhaps, that cancer continues to be very rare among hunter-gatherer societies which derive the majority of their calories from animal fat and protein, with only a small contribution from complex carbohydrates such as berries and roots [15, 16]. These observations, along with an increasing understanding of cancer metabolism, have prompted research into potential dietary therapies for the prevention and treatment of cancer. The ketogenic diet (KD) is a high fat, adequate protein, very low carbohydrate diet, classically consisting of a 4:1 macronutrient ratio of fats: protein + carbohydrate. It is most wellcharacterized clinically for its use in treating pediatric refractory epilepsy [17], but is proposed to have therapeutic efficacy for a variety of disorders, including obesity, diabetes, hypertension, Amyotrophic Lateral Sclerosis (ALS), Alzheimer s, and Parkinson s disease [18-20]. Like cancer, many of these diseases exhibit underlying metabolic pathology [21, 22] and chronic inflammation thought to be associated with a state of hyperglycemia [23-25]. The KD mimics a metabolic state of starvation, inducing a physiologic shift towards fat metabolism and a state of ketosis. The carbohydrate restriction of the KD keeps glucose and insulin levels low, activating 46

64 gluconeogenesis, and forcing the acetyl CoA produced from fatty acid β-oxidation to be shunted towards ketogenesis in the liver. Decreasing circulating blood glucose with the KD is one method of exploiting the Warburg effect and is thought to selectively starve growing tumors of the glycolytic substrates which feed their rapid proliferation [26]. Although glucose deprivation was the original intent of using the KD as a cancer treatment, further investigation has uncovered many other mechanisms which may underlie the anti-cancer effects of this therapy Literature Review The KD has been shown to slow cancer progression in a small number of animal and human studies for a variety of cancer types. In the 23132/87 xenograft model of human gastric adenocarcinoma, a KD supplemented with medium chain triglycerides (MCTs) and omega-3 fatty acids fed ad libitum significantly slowed tumor growth and increased survival time [27]. Tumors in the KD-fed mice were less vascularized and had more necrotic areas than standard diet (SD) fed mice. In in the LAPC-4 xenograft model of prostate cancer, the KD slowed tumor growth but did not increase survival time [28, 29]. However, in the LNCaP xenograft model of prostate cancer, the KD significantly prolonged survival [30]. Seyfried and colleagues have published many reports on the calorie restricted ketogenic diet (R-KD) as a therapy for brain cancer. The R-KD decreased tumor weight when administered as a stand-alone therapy in the CT-2A malignant mouse astrocytoma model; however, it elicited potent synergistic anti-cancer effects when administered in conjunction with the glycolytic inhibitor 2-deoxy-D-glucose (2- DG) [31]. In the same model, Seyfried et al showed that blood glucose levels had a direct and 47

65 positive correlation with tumor growth and that the R-KD significantly decreased plasma glucose [32]. The R-KD also decreased plasma insulin-like growth factor-1 (IGF-1) levels, a known biomarker for cancer progression and angiogenesis. Similar results were demonstrated by Mukherjee et al wherein the R-KD decreased proliferation and vascularization and increased apoptosis in two prostate cancer models in vivo [33]. As was also observed by Seyfried s group, calorie restriction of both a standard and KD elicited similar therapeutic effects in these models, suggesting that for some cancers total energy restriction may be a more important component of dietary therapy than macronutrient profile of the diet. The KD has been evaluated in other brain tumor models as well. Scheck and colleagues showed a significant synergistic response to the combination of KD and radiation therapy in the GL261 mouse model of glioma [34]. The KD fed ad libitum increased survival time by approximately 22%, but when administered concomitantly with radiation therapy, 82% of mice exhibited complete and permanent tumor remission, even after returning to a standard diet. Similar synergistic effects were seen in two lung xenograft models, with the KD enhancing the efficacy of both radiation and carboplatin chemotherapy [35]. The earliest report of using the KD as a cancer therapy in humans was published by Nebeling et al in 1995 [36]. This study was designed to determine if the KD could decrease glucose uptake by tumors in two female pediatric patients with late stage malignant astrocytomas. Glucose uptake by FDG-PET imaging was decreased by an average of 21.8% in the two patients. One patient responded remarkably well to dietary therapy, exhibiting clinical improvement in quality of life and motor function. At the time of the report, she had continued the KD therapy for an additional year with no disease progression. Zuccoli et al published a case report on a 65 year old woman with multicentric glioblastoma multiforme (GBM) treated with a 48

66 calorie-restricted ketogenic diet given concomitantly with standard care [37]. The patient responded well to dietary therapy, and after two months of treatment, her tumors were no longer visible by FDG-PET or MRI imaging. Ten weeks after discontinuing the dietary therapy, however, her tumors returned and she eventually succumbed to the disease. In recent years, a few small clinical trials have evaluated the effects of the KD on patients with late-stage, metastatic cancer. In one such study, sixteen patients with advanced metastatic cancers were placed on a KD for 3 months and monitored for changes in quality of life and general health parameters [38]. The patients who completed the dietary regimen exhibited improvement in some factors pertaining to quality of life, such as better emotional functioning and decreased insomnia, with no significant adverse side effects. Fine and colleagues recently reported the effects of what was described as an insulin inhibiting diet, but was essentially a very low carbohydrate ketogenic diet, in a group of 10 patients with advanced metastatic disease of a variety of tissue origins [39]. Although all patients had previously exhibited rapid disease progression, five of the nine patients who completed the study showed stable disease or partial remission during the 28 day dietary therapy. A review of the literature strongly supports the potential use of the KD as an anti-cancer therapy; however, further validation in preclinical and clinical studies is needed to fully understand its use Anti-Cancer Effects of the Ketogenic Diet Decreased Glucose and Insulin Signaling Due to the Warburg effect, glucose from dietary carbohydrate provides the majority of metabolic fuel to most, if not all, cancers. This observation prompted initial research into the 49

67 KD as a cancer therapy, with carbohydrate restriction-induced glucose deprivation widely regarded as the major mechanism by which the KD slows tumor progression. It is well established that tumor growth is directly correlated with blood glucose levels and that hyperglycemia increases tumor growth rate in animals and in humans [32, 40, 41]. Glucose uptake is a rate-limiting step of glucose metabolism and is mediated by the glucose transporter (GLUT) proteins. GLUT expression is consistently elevated in cancer cells of varying tissue origins [42]. Of the 14 known GLUT isoforms, GLUT 1 and GLUT 3 overexpression is most often associated with malignant transformation and progression, and both have been correlated to poor prognosis clinically [42]. The low Km of these enzymes allow tumors to import glucose from the blood even at relatively low plasma concentrations [43]. Excess glucose availability provides the glycolytic substrates to fuel the Warburg effect and all of its aforementioned beneficial consequences. It also provides an energy substrate to support cellular functions despite the mitochondrial dysfunction which is so often associated with cancers [44]. Therefore, the KD is thought to work in large part by decreasing glucose availability to the tumor. As described, the KD has been shown to decrease glucose uptake by tumors in humans [36]. Some studies suggest that the KD given in unrestricted amounts does not decrease blood glucose [27, 32], while others reported a significant drop in glucose levels with the KD even when fed ad libitum [34, 45]. This variability is likely due to the individual heterogeneity of metabolism which is becoming increasingly more-well understood [46], as well as differences in the macronutrient sources in the respective KDs studied. Hyperglycemia stimulates insulin secretion from the pancreas. High levels of circulating insulin, or hyperinsulinemia, is also associated with an increased risk of colorectal, pancreatic, and breast cancer, among others [47]. Hyperinsulinemia is closely related to obesity and 50

68 diabetes, both of which are known risk factors for cancer development. However, studies suggest that hyperinsulinemia increases the risk of cancer mortality independently of diabetes, obesity, or metabolic syndrome [48, 49]. Insulin binds to the insulin receptor (IR) to mediate the cellular effects of glucose metabolism, but it also exerts a potent growth factor response. IR expression and signaling is commonly over-activated in cancers and is considered to play an important role in promoting tumor growth and progression [50]. IR activation stimulates Ras and the MAPK cascade which mediate the mitogenic effects of insulin, promoting cell proliferation. IR also works through the PI3K pathway to promote cell survival through Akt and mtor. Furthermore, Akt promotes expression of NFκB which activates pro-inflammatory and anti-apoptotic programs. Growth promotion by insulin signaling cross-talks with the β- catenin/wnt pathway, and therefore may play a role in dedifferentiation and malignant progression [51]. Insulin also induces vascular endothelial growth factor (VEGF) expression, a potent activator of angiogenesis [52, 53]. Angiogenesis is required for tumor growth and plays a critical role in cancer progression and metastasis. The KD decreases pancreatic insulin production by restricting carbohydrate intake. It has also been shown to enhance insulin sensitivity in healthy tissues, which leads to decreased circulating insulin in the blood [54]. The KD has been reported to reduce vascularization in a xenograft model of gastric cancer, possibly through its inhibition of insulin signaling [27]. In a mouse model of prostate cancer, a reduction in tumor growth rate and prolonged survival was associated with a decrease in serum insulin in mice fed the KD [55]. In a small human trial, patients with end-stage cancer receiving a KD for 28 days exhibited a significant drop in blood insulin [39]. This decrease in insulin was inversely correlated to the degree of relative ketosis, which was positively correlated to therapeutic response [39]. Therefore, the KD may work in part by inhibiting insulin signaling in tumors. 51

69 Reduced Flux through the Pentose Phosphate Pathway A major consequence of the Warburg effect is increased flux through the pentose phosphate pathway (PPP). As described, the PPP generates ribose-5-phosphate and NADPH, biosynthetic precursors necessary for nucleotide and lipid synthesis as well as endogenous antioxidant regeneration. By decreasing glucose, the glycolytic substrate, the KD will decrease flux through the pentose phosphate pathway (PPP). This will limit production of the anabolic substrates necessary for cell division and inhibit proliferation of the growing tumor. Since the PPP also contributes reducing equivalents in the form of NADPH, up-regulation of this pathway provides a protective advantage to the cancer cell against radiation and chemotherapy insults which act through ROS-induced oxidative damage. Intracellular levels of reduced glutathione (GSH) and ROS are tightly linked to regulation of cell death, suggesting that overstimulation of the PPP in cancer cells plays a role in evasion of apoptosis. Thus, reducing flux through the PPP with the KD may also enhance the efficacy of standard care and sensitize the cancer cells to cell death. Furthermore, two key enzymes of the PPP glucose-6-phosphate dehydrogenase (G6PD) and transketolase-like 1 (TKTL) are now widely accepted as oncogenes [56, 57], leading some to propose that dysregulation of the PPP should be considered a hallmark of cancer [58]. G6PD and TKTL are often overexpressed in cancer, and both are linked to tumorigenesis and cancer progression [56, 57]. The KD may also inhibit cancer progression by decreasing the activity of these two enzymes. 52

70 Influence on ROS Production and Redox Status There appears to be a significant differential effect of the KD on cancer versus normal tissue, possibly due to mitochondrial damage in the cancer cells. In healthy tissues, the KD protects against oxidative stress by simultaneously decreasing ROS production and enhancing endogenous antioxidant capacity. As described, the KD induces a physiological shift away from carbohydrate metabolism and towards fat and ketone metabolism. Veech and colleagues studied the effects of ketone metabolism by investigating energy efficiency and mitochondrial respiration after administering a glucose-containing perfusate supplemented with 5mM βhb in a working rat heart [59]. Their studies showed that ketone metabolism increases the oxidation of co-enzyme Q in the electron transport chain, thus reducing production of the superoxide anion (O2 - ), an important precursor for the generation of many ROS [59]. Ketone metabolism also caused a reduction of the mitochondrial NAD and cytoplasmic NADP couples which are necessary for regenerating reduced glutathione (GSH). These effects appear to be ubiquitous and have been confirmed by studies in other tissues, most notably the brain. βhb and AcAc decrease neuronal ROS production in vitro in response to glutamate [60] and prevent cell death in cortical slices following hydrogen peroxide (H2O2) exposure [61]. When fed for 3 weeks, the KD increased the ratio of reduced to oxidized glutathione (GSH:GSSG) in rat hippocampi, suggesting an improvement in antioxidant capacity [62]. These mechanisms likely underlie many of the well-known neuroprotective effects of the KD which have been reported in a number of pathologies, like Alzheimer s disease, Parkinson s disease, ALS, and epilepsy, among others [63]. As described, the role of ROS in cancer is complex, with tumors benefitting from basally elevated ROS production yet sensitive to even modest changes in redox state [64]. 53

71 Consequently, researchers are investigating both pro-oxidant and anti-oxidant treatments as potential cancer therapies. Radiation and many chemotherapies work in large part by inducing ROS production while exogenous antioxidant supplements, such as vitamin C, are thought to work by quenching the ROS produced by tumors [65]. While the KD clearly elicits an antioxidant response in healthy tissues, its effects on cancer is unclear. Scheck and colleagues showed that, similar to its effects in healthy brain tissue, the KD reduced ROS production and enhanced endogenous antioxidant expression in a mouse model of glioma [66]. Fath et al examined the effects of the KD, radiation, and carboplatin chemotherapy on oxidative stress in two lung cancer xenograft models [35]. They showed that the animals fed a KD in combination with radiation therapy exhibited increased lipid peroxidation and oxidative damage. The differential effects of the KD on healthy versus cancer tissue remain to be elucidated but may depend in part on the tissue of origin and/or mitochondrial damage in the tumors [44]. Taken together, these studies suggest that the KD may work in part by increasing ROS production in the tumor while simultaneously protecting against oxidative stress in healthy tissues. If this is the case, combining the KD with radiation or chemotherapy may enhance the efficacy of standard care while decreasing adverse side effects in patients Inhibition of Inflammation It is now widely accepted that chronic inflammation plays a critical role in tumorigenesis [67]. In their 2011 updated review, Hanahan and Weinberg added tumor promoting inflammation alongside genome instability and mutation as enabling characteristics which underlie the hallmarks of cancer [68]. Growing evidence suggests that up to 15% of all cancers 54

72 worldwide may be attributed to persistent infections [69]. Infiltrating immune cells mount an inflammatory response largely mediated by ROS which oxidize the nuclear genome of host cells. This DNA damage contributes to the accumulation of oncogene and tumor suppressor gene mutations which mediate malignant transformation. Once a tumor forms, pro-inflammatory cytokines released from tumor itself and immune cells in the microenvironment foster a physiological niche which promotes cancer progression, invasion, and metastasis [67]. TNF-α, TGF-β, IL-6, IL-8, IL-10, CXCL1-3, and MIP-3 are some of the cytokines shown to mediate angiogenesis, tumor growth, invasion, and metastasis in a variety of cancers [67, 70]. The KD may inhibit progression and induce cell death in cancers in part by inhibiting inflammation. By comparing the effects of isocaloric high carbohydrate, low fat diet versus very low carbohydrate KDs, studies have shown that the KD reduces circulating inflammatory markers in humans [71]. In the study, patients on the KD exhibited decreases in numerous proinflammatory cytokines, such as TNF-α, IL-6, IL-8, MCP-1, E-selectin, I-CAM, and PAI-1 [71]. A similar study by the Volek lab showed a significant decrease in TNF-α, IL-6, C-reactive protein (CRP), and ICAM-1 following weight loss induced by the KD [72]. A study in guinea pigs showed that the KD reduced circulating levels of all of the 15 pro-inflammatory cytokines tested, including IL-1β, IL-2, IL-4, IL-7, IL-9, IL-12, IL-13, IL-15, IFN-γ, GM-CSF, and RANTES, among others [73]. Although the effect of the KD on inflammation in the tumor microenvironment remains largely untested, it is logical to hypothesize that its anti-inflammatory effects would persist there as well. Therefore, the KD could inhibit cancer progression in part by reducing inflammatory signaling. Furthermore, hyperglycemia is also known to be a major cause of chronic inflammation and oxidative stress [74]. This observation elucidates another link 55

73 between altered metabolism, tumorigenesis, and cancer progression as well as another mechanism by which the KD may inhibit tumor growth Potential Concerns or Side Effects Variability in the Effects of the Ketogenic Diet It is important to acknowledge that the scientific literature investigating the effects of the KD includes a wide variability in dietary design and therapeutic outcomes. Some studies, especially in animals, have reported anti-cancer effects of the KD only when administered in calorie restricted amounts [32, 75, 76]. These studies have also indicated that if over-consumed, the KD can lead to hyperlipidemia and insulin insensitivity, and is not effective at lowering blood glucose [32, 77, 78]. Other studies in animals and humans have reported anti-cancer and other beneficial effects of the KD without significant adverse effects, even when consumed ad libitum [34, 45, 54, 66, 79-82]. It is possible that certain cancers will be more susceptible to KD therapy only when administered in calorically restricted amounts. However, it is also very likely that variability in the macronutrient composition of the specific KD studied plays a significant role in these differential effects. KDs that are well-balanced, such as those composed largely of ketogenic medium chain triglycerides, contain a healthy mixture of saturated and monounsaturated fats, and possess a low ratio of omega-6 to omega-3 polyunsaturated fatty acids (PUFAs), will likely provide more benefits with less adverse effects than unbalanced KDs, such as those composed almost entirely of lard or vegetable oil. Furthermore, the appetite suppressing effects of the KD typically prevents overconsumption in humans, and is often unintentionally accompanied by calorie restriction which contributes to decreased blood glucose and other 56

74 beneficial effects [83, 84]. Therefore, it is important to recognize the caveats of these dietary studies and to use well-formulated KDs to evaluate new therapeutic potential Cancer Cachexia and Weight Loss Currently, very little information is provided to patients regarding optimal dietary guidelines following a cancer diagnosis. Of the 21 National Comprehensive Cancer Network (NCCN) member institutions identified on only 4 of the institutions websites provide any nutritional guidelines for patients at all [85]. Rather than considering the effect of diet on cancer progression itself, the primary concern of diet for cancer patients clinically involves avoiding body weight loss associated with cancer cachexia. As such, the most commonly recommended diets are low-fat, high-carbohydrate diets consisting of a 5:1 or 7:1 ratio of carbohydrate to fat with high calorie intake [85]. Considering the effect of blood glucose on tumor growth, these dietary recommendations may be inadvertently promoting cancer progression. However, the KD is a well-known weight loss tool for overweight individuals [86] and therefore it is important to consider its effects on body weight and muscle mass. Cachexia is generally defined as weight loss and is characterized by loss of muscle mass, with or without loss of fat mass [87]. It is a consistent feature of late-stage cancer, with 86% of patients experiencing weight loss by the last 2 weeks of life [88]. While fat mass often decreases, the major detriment to health from cancer cachexia comes from loss of muscle mass. Up to 20% of cancer deaths are caused by cachexia-induced cardiac or respiratory failure [89]. In fed conditions and while eating a high carbohydrate diet, the brain derives nearly all of its energy from glucose. Glucose availability from glycogen stores are limited, however, and can 57

75 only satisfy the cerebral energy requirement for a short period of time without food before requiring additional glucose produced by gluconeogenesis of amino acids from muscle protein degradation [90]. If the brain was entirely reliant on glucose, starvation-induced muscle proteolysis would quickly interfere with cardiac and respiratory function and become fatal within 2-3 weeks. Therefore, the human brain evolved to metabolize ketones produced by the liver from stored fats during periods of calorie deprivation or starvation in order to spare muscle protein [90, 91]. This adaptation allows most adult humans to prolong survival during starvation to two or more months, depending on the amount of storage fat (triglycerides). For this reason, ketones are considered to elicit a protein sparing effect [92]. While the KD promotes weight loss in obese individuals, it has been shown to prevent muscle loss during physical stress or energy restriction [93, 94]. Elevated blood ketones inhibit amino acid release from muscles as well as hepatic gluconeogenesis [95]. It makes sense that the KD would elicit similar protective effects against cancer cachexia. Indeed, Fearon and colleagues reported that patients with late-stage cancer and cachexia fed a ketone-supplemented KD maintained a positive nitrogen balance and regained some of their lost weight [96]. In a mouse model of cachexia, animals fed the KD had smaller tumors but did not lose as much body weight compared to controls [97]. KD-fed animals also had lower levels of circulating catabolic factors, which stimulate lipolysis and proteolysis, than controls. In similar studies, animals with colon cancer-induced cachexia showed a significant up-regulation of the ketone utilization enzyme 3-oxoacid CoA-transferase, suggesting a greater reliance on ketones for energy [98]. Supplying ketones to the body with the KD could support the health and metabolic needs of these tissues to withstand the physiologic stress of cachexia. These studies indicate that the KD will not negatively affect body weight or muscle mass in cancer patients, but may actually prevent or reverse the cachexic phenotype. 58

76 Effects on Cardiovascular Health There is a common belief held by the medical and general populations that consuming large amounts of dietary fat is unhealthy and will lead to heart disease and increased mortality. Unfortunately, the history of nutritional science and guidelines is fraught with poor science and political interference [99]. Scientific evidence supporting the fat-heart hypothesis has always been controversial, and new, well-designed studies and meta-analyses of the literature are challenging the current dogma. Recently, a systematic review and meta-analysis of over 75 studies concluded that the current body of evidence does not support the common cardiovascular guidelines regarding fat consumption and found no significant correlation between risk of heart disease and fat intake [100]. In fact, isocaloric studies comparing low fat, high carbohydrate to well-formulated very low carbohydrate ketogenic diets (VLCKD) in humans show that the VLCKD improves biomarkers of cardiovascular health by decreasing body weight, decreasing trunk fat mass, improving body composition, decreased blood glucose and insulin, decreased blood triglycerides, and improved cholesterol ratio [79, ]. In follow-up studies of children who followed the KD for several years to manage their retractable epilepsy, researchers found no significant long-term side effects associated with the diet [17, 104]. These studies suggest that there are no significant risks associated with the KD on cardiovascular or general health and can be considered a potential non-toxic therapy for cancer patients. 2.3 Targeting Cancer Metabolism with Ketone Supplementation Unlike normal tissues, many cancers do not seem capable of efficiently metabolizing ketone bodies for energy. Cancer cells often lack expression of the ketone utilization enzymes, 59

77 like succinyl-coenzyme A:3-oxoacid coenzyme A transferase (SCOT) [ ]. A study on 5 different glioma cell lines concluded that glioma cells are unable to metabolize βhb to compensate for glucose restriction like healthy brain cells [109, 110]. Furthermore, as ketones are metabolized exclusively within the mitochondria, most tumors would have a reduced ability to use ketones for energy due to their dysfunctional mitochondria and impaired OXPHOS capability. The ketogenic diet, fasting, and calorie restriction are dietary regimens that have been shown to inhibit cancer progression in both preclinical and clinical studies [30, 36, 37, 39, 45, 55, ]. The generally accepted mechanism by which these therapies may work is by decreasing glucose availability to the tumor and suppressing the insulin and IGF signaling pathways. However, all three of these therapies also induce a state of elevated blood ketones, or ketosis. A recent study examined the effects of 28-day KD in patients with late-stage, metastatic cancers of a variety of tissue origins, including breast, fallopian tube, colorectal, lung, esophageal, ovarian, and lung [39]. All patients had prior progressive disease confirmed by FDG-PET upon beginning the dietary intervention. After the one month treatment, over fifty percent of patients showed stable disease or partial remission. Interestingly, there was no significant drop in blood glucose in the patients over the course of the diet. Upon further analysis, the researchers determined that patient response was most strongly correlated with degree of relative ketosis from baseline. This study was not the first, however, to hint at the anticancer effects of ketones. In 1979, Magee et al demonstrated that βhb inhibited proliferation in a dose dependent manner up to 20 mm in transformed lymphoblast, HeLa, and melanoma cell lines [114]. A more recent study showed that both AcAc and βhb inhibited viability and 60

78 induced apoptosis in neuroblastoma cells, but had no effect on control fibroblasts [105]. These observations strongly suggest that ketone bodies may possess inherent anti-cancer properties Exogenous Ketone Supplementation Ketones are naturally produced in the body only under certain physiological conditions, including starvation, fasting, prolonged exercise, or during the consumption of a carbohydrate restricted ketogenic diet. However, it is possible to elevate blood ketones artificially with sources of exogenous ketones or ketogenic agents. These agents serve as ketogenic precursors and are either enzymatically cleaved or metabolized to produce ketones. Thus, ketone supplementation can elevate blood ketone levels without the need for severe dietary restrictions. It is possible that exogenous ketone supplementation could provide many of the therapeutic effects associated with the KD without the need for severe dietary restriction. It is also very likely that a ketone supplemented ketogenic diet would be easier to maintain and a more efficacious therapy than a standard KD or ketone supplementation alone. 1,3-butanediol (BD) and ketone esters are two sources of ketone supplements which can be administered to elevate blood ketones exogenously , 3-Butanediol 1, 3-Butanediol (BD) is an organic alcohol that was investigated in the 1950s as a calorie dense food for extended space travel [115]. After an adaptation period, BD contributes approximately 6 kcal/g of energy. Extensive toxicology analyses indicate that BD is safe, with 61

79 very low acute and chronic toxicity and no teratogenic effects [115, 116]. Today it is FDA approved and most commonly used as a food flavoring solvent. When orally administered, BD is metabolized by the liver to produce milimolar levels of βhb and can therefore be used as an exogenous source of ketone supplementation [117, 118] Ketone Esters In attempts to harness the therapeutic potential of the KD without severe dietary restrictions, researchers have developed synthetic ketone esters which are metabolized to the ketone bodies in vivo. Birkhahn et al were the first to provide a practical method of administering large quantities of ketone bodies without sodium overload using a monoester of glycerol and AcAc [ ]. Henri Brunengraber and Richard Veech have also developed a number of ketone esters which have been shown to elevate blood ketones in vivo [ ], and toxicity studies indicate that these ketone esters are safe [126, 127]. The R, S-1, 3-butanediol acetoacetate diester (KE) is a nonionized, water-soluble precursor to ketone bodies designed by Dr. Dominic D Agostino and created for our lab in collaboration with Patrick Arnold of Savind Inc. Gastric esterases rapidly catalyze the release of AcAc, liberating R, S-1, 3-butanediol which is metabolized by the liver to produce βhb [128, 129]. Thus, the KD causes a rapid elevation in blood AcAc followed by a more sustained production of βhb. Single dose administration of the KE via intragastric gavage raised ketone levels to therapeutic levels (> 3mM βhb and >3 mm AcAc) in rats, and mimicked the anti-convulsant effects of the KD by increasing latency to oxygen toxicity-induced seizures [128]. Thus, the KE is a good source of exogenous ketone supplementation to investigate as a potential cancer therapeutic. 62

80 2.3.2 Anti-Cancer Effects of Ketone Supplementation Inhibition of Glycolysis Ketone bodies are known to have a regulatory effect on glycolysis and glucose metabolism. Early studies demonstrated that βhb inhibits glucose uptake by hearts of fed rats with normal glucose concentrations [130]. Furthermore, βhb inhibits the first and third enzymatic reactions of glycolysis, the phosphorylation of glucose by hexokinase and the phosphorylation of fructose-6-phosphate by phosphofructokinase [130]. In a study in fastedchick muscles, ketone bodies did not decrease glucose uptake but did significantly inhibit glycolysis and decrease intracellular pyruvate production [92]. Thus, ketone supplementation could directly inhibit the glycolytic enzymes of cancer cells which are heavily reliant on this metabolic pathway Influence on ROS Production and Redox Status The effect of ketone supplementation on ROS and redox status in cancer versus healthy tissue would likely be very similar to the previously described effects of the KD (Section ). Indeed, the antioxidant effects of the KD are largely attributed to ketone metabolism, which is known to decrease ROS production and increase endogenous antioxidant capacity in healthy cells [63, 95]. However, the inability of cancer cells to efficiently metabolize ketones for energy brings to question what effect ketones would have on redox state in tumors. Electron transport is impeded in damaged mitochondria, causing them to produce more ROS than healthy mitochondria [64]. Ketone supplementation could potentially enhance this ROS production by shuttling more electrons through the ETC of damaged cancer mitochondria. Dichloroacetate 63

81 (DCA) is a small molecule pyruvate mimetic that inhibits pyruvate dehydrogenase kinase, the inhibitor of pyruvate dehydrogenase complex, thus stimulating pyruvate conversion to acetyl CoA and entry into the Kreb s cycle [131]. It is generally considered a stimulator of mitochondrial OXPHOS and is being investigated for its use as a cancer therapeutic. DCA increases mitochondrial ROS production to a greater extent in cancer cells than healthy cells, and this discrepancy is tied to defects in mitochondrial ETC [131]. This observation supports the idea that shuttling energy metabolites through the ETC of dysfunctional mitochondria may damage cancer cells by increasing ROS production. Ketone supplementation may impair cancer in this way. Alternatively, ketone supplementation could elicit an antioxidant effect in cancer cells, similar to its effect in normal cells [60]. The response will likely be dependent on the degree of mitochondrial dysfunction in the particular tumor. However, as cancers are susceptible to both pro-oxidant and anti-oxidant treatments, it is possible that either effect could provide therapeutic potential Histone Deacetylase Inhibitor Activity The role of ketones as an energy metabolite has been long known; however, emerging evidence suggests that they have important functions as signaling molecules as well [132]. Histone methylation and acetylation are major regulatory mechanisms which modulate DNA expression. Cancers exhibit widespread differences in such epigenetic patterns compared to their normal tissue counterparts, allowing them to increase expression of oncogenes and inhibit the expression of tumor suppressor genes [133]. Histone deacetylases (HDACs) are a family of enzymes which catalyze the removal of acetyl groups from DNA histones and therefore play 64

82 critical roles in mediating these gene expression changes. As such, histone deacetylase inhibitors (HDACI) have been investigated for their use as antineotplastic agents. Preclinical evidence suggests that HDACIs may be efficacious in a number of malignancies, including acute myeloid leukemia (AML) and multiple myeloma (MM) [134, 135]. There are currently two HDACIs approved by the FDA for the treatment of T-cell lymphoma: vorinostat and romidpesin [136]. HDACIs elicit a plethora of anti-cancer effects in vitro including inhibition of anti-apoptotic proteins, activation of pro-apoptotic proteins, induction of ROS generation and DNA damage, and inhibition of DNA repair [137]. Verdin and colleagues recently demonstrated that βhb acts as an endogenous HDACI both in vitro and in vivo at milimolar levels easily achievable with the KD or ketone supplementation [138]. Many of the genes upregulated by βhb were involved in protection against oxidative stress, including Foxo3a, a transcription factor which induces cell-cycle arrest. It is possible that ketone supplementation could elicit similar effects as the pharmaceutical HDACIs in cancer cells. Additionally, although histones were the first characterized targets of HDACIs, other non-histone proteins are also deacetylated by HDACs, including p53 and c-myc [139]. This suggests that ketones could directly modulate the expression of important oncogenes and tumor suppressor genes Enhancement of Mitochondrial Health and Function There is significant evidence to suggest that healthy mitochondria suppress the tumor phenotype. As the name suggests, nuclear-cytoplasmic transfer studies involve the transfer of nuclei from one cell into the cytoplasm of another. Several groups have investigated how tumor 65

83 cell nuclei behave when placed in cytoplasm from healthy cells, and vice versa. Cybrid studies on melanoma cells indicated that the cytoplasm from healthy myoblasts suppressed their tumorigenicity in vitro [140]. Similarly, the cytoplasm from normal hepatocytes inhibited tumorigenicity of transformed hepatocytes, reducing in vivo tumor formation by 40% [141]. The same group showed in similar studies that while cybrids containing nuclei derived from malignant cells and cytoplasm from healthy cells formed tumors in only 17% of animals tested, cybrids containing cytoplasm from malignant cells and nuclei from healthy cells formed tumors in 97% of animals [142]. More recently developed techniques allow for the specific transfer of isolated mitochondria between cells. These studies have demonstrated very similar effects. Mitochondrial transfer from normal mammary epithelial cells into a malignant osteosarcoma cell line restored respiratory function, inhibited proliferation and viability, induced pro-apoptotic signaling and sensitivity to chemotherapy, and inhibited invasive and colony formation properties in vitro [143]. These cybrids also showed reduced tumor growth in nude mice. Many other nuclear-cytoplasmic or mitochondrial transfer studies have reported similar findings in a variety of cancer cell types [ ]. Taken together, these studies strongly suggest that the mitochondria play a much more significant role in tumorigenesis than is currently acknowledged, and should be incorporated into the currently accepted theory which is decidedly genocentric [151]. It is also clear that healthy mitochondria can suppress the neoplastic phenotype, likely in part by maintaining ATP production which is critical for DNA repair and preservation of the integrity of the genome [152, 153]. Therefore, therapies which enhance mitochondrial health or function could prevent carcinogenesis and/or inhibit cancer progression. Ketone metabolism is generally recognized to support mitochondrial health [95]. As described, ketones reduce mitochondrial ROS and enhance endogenous antioxidant defense mechanisms. Therefore, they 66

84 can protect the mitochondrial membranes and genome from oxidative damage which impair respiratory and mitochondrial function. Furthermore, Veech and colleagues have demonstrated that dietary ketone supplementation with a ketone ester induces mitochondrial biogenesis [125]. Mitochondrial biogenesis in tumors with low mitochondrial numbers, as many exhibit, may restore some degree of normal mitochondrial function. Indeed, mitochondrial amplification was shown to increase breast cancer chemosensitivity to doxorubicin [154], suggesting that mitochondrial biogenesis could also enhance the efficacy of standard care. It is possible that ketone supplementation could inhibit cancer progression by enhancing mitochondrial biogenesis or improving mitochondrial health and function Inhibition of Lactate Export Both lactate and the ketone bodies are transported across the plasma membrane by the MCT family of transporters [155]. As previously described, inhibiting lactate export from cancer cells has a negative effect on survival and proliferation [156]. βhb has been shown to inhibit lactate export from isolated rat hepatocytes in vitro [157]. It is possible that ketones may damage cancer cells by inhibiting lactate export through competitive inhibition of MCTs, subsequently inducing intracellular acidification and preventing the tumor promoting effects of lactate in the tumor microenvironment. 67

85 2.3.3 Potential Concerns or Side Effects Ketoacidosis and Effect on Blood ph Because of the macronutrient profile and calorie consumption of the standard Western diet, ketone bodies are not often detectable in the blood of healthy adults. However, they make a dangerous appearance in patients with uncontrolled diabetes. In diabetic ketoacidosis (DKA), high blood glucose levels and dysregulated insulin signaling induces unrestrained ketogenesis. Blood ketone levels may rise to the range of mm. Since ketones are acids, at these elevated levels, blood ph drops and can cause widespread organ failure. If not properly administered, ketone supplementation has the potential to induce ketoacidosis. However, we anticipate that ketone supplementation could inhibit cancer progression within a range of therapeutic ketosis (2 7 mm), levels far below the range at which blood ph is affected. 2.4 Targeting Cancer Metabolism with Hyperbaric Oxygen Therapy Hyperbaric oxygen therapy (HBOT) is a treatment in which patients breath 100% oxygen in a hyperbaric chamber which has been pressurized to greater than the barometric pressure of sea level, 1 atmosphere absolute (ATA). HBOT can be administered in mono-place (single person) or multi-place (multi-person) chambers which are typically pressurized between 2 to 3 ATA for 1.5 to 2 hours at a time [158]. While mono-place chambers can be pressurized with 100% oxygen, multi-place chambers are often pressurized with air while the patients inside breathe 100% oxygen through a mask or endotracheal tube. The Undersea and Hyperbaric Medical Society (UHMS) has approved the use of HBOT for a variety of disorders, including decompression sickness, carbon monoxide poisoning, and radionecrosis among others [159]. 68

86 The physiological basis of HBOT as a treatment modality is based on the gas laws. Dalton s law states that in a mixed gas, each element exerts a pressure proportional to its partial pressure, or its fraction of the total volume. Henry s law states that the amount of gas dissolved in a liquid or tissue is proportional to the partial pressure of that gas in contact with the liquid or tissue. While breathing air at atmospheric pressure, most of the oxygen carried through the blood is bound to hemoglobin. In this situation, blood hemoglobin is working at nearly maximum capacity and is approximately 97% saturated with oxygen, with only a small amount of oxygen dissolved in the blood plasma compartment. According to Dalton s and Henry s laws, by increasing the percent of oxygen breathed, or the pressure at which oxygen is breathed, more oxygen will dissolve into the blood plasma. HBOT increases both the percent and pressure of oxygen breathed, supersaturating the blood with oxygen. The increase in blood oxygenation following HBOT can be significant. Breathing 100% oxygen at 3 ATA increases mean arterial oxygen tension from approximately 100 mm Hg to 2000 mm Hg and mean tissue oxygen tension from approximately 55 mm Hg to 500 mm Hg [160]. This level of HBO increases the amount of oxygen delivery to the tissues by 20-fold, permitting the provision of 60 ml of oxygen per liter of blood compared to the 3 ml/l which is delivered while breathing normobaric air [161]. Supersaturating the blood with oxygen to this degree can adequately support the resting tissue oxygen requirement without a contribution from hemoglobin carriage, making HBO an ideal therapy for diseases in which hemoglobin function is impaired, such as anemia or carbon monoxide poisoning [162]. Because the excess oxygen is carried in solution, it can diffuse farther into the tissues to maximize tissue oxygenation, even spreading to areas where red blood cells cannot reach. 69

87 2.4.1 Literature Review The potential benefits of HBOT as a cancer therapy are clear. Since tumor hypoxia promotes the cancer phenotype through several mechanisms, saturating solid tumors with HBOT may be able to reverse many of the cancer-promoting effects of hypoxia. Indeed, HBOT has been shown to slow cancer progression in a variety of animal and human cancers. Comprehensive reviews on the topic conclude that overall, HBOT most often elicits a cancerinhibiting or neutral effect, with only a very small number of studies reporting a cancerpromoting effect [ ]. There is significant variability in response between in vitro, in vivo, and clinical studies on HBOT and cancer, with multiple studies reporting positive or neutral responses in tumors of the same tissue of origin. It is also sometimes found to only be effective when given as an adjuvant to radiation or chemotherapy, but not when administered alone. Other studies report a positive effect of HBOT as a stand-alone treatment, suggesting the therapy efficacy may be highly tissue and context dependent. Stuhr and colleagues have published several studies using clinically-relevant HBOT protocols which induced a significant inhibition on tumor growth in mouse models of breast cancer [ ]. Two small studies in humans did not report an effect of HBOT on cancer progression or survival [170], but did report decreased pain, edema, and erythema following radiation breast-conserving surgery [171]. HBOT improved the outcome of patients with colorectal cancer when given as an adjuvant to radiation therapy [172], and animal studies suggest that it may only be effective when given as an adjuvant to radiation, photodynamic therapy, or chemotherapy for this cancer type [173, 174]. Gliomas appear to be a cancer type where HBOT may be effective to some degree as a stand-alone therapy. Increasing po2 in a rat xenograft model with both normobaric hyperoxia and low-level HBO reduced tumor 70

88 growth and vascular density [175]. However, it is quite possible the response could vary with an orthotopic implantation model. Kohshi and colleagues reported that HBOT given as an adjuvant to radiotherapy in patients with malignant gliomas increased mean survival time from 12 to 24 months compared to patients receiving radiotherapy alone [176]. Another study demonstrated that HBOT prolonged the biological residence time of the chemotherapy carboplatin in patients with malignant glioma [177]. In vitro studies showed that HBOT decreased proliferation, induces apoptosis, and sensitized prostate cancer cells to chemotherapy treatment [178, 179]; however, animal studies in models of prostate cancer found no significant effect [ ]. In vitro studies have shown that HBOT induces apoptosis in several different leukemia cell lines [183, 184], suggesting it may be effective against this malignancy as well [185]. However, it has not been thoroughly investigated in animals or humans. The studies described here represent some of the most recent work done investigating HBOT and cancer [164], but fifty years of prior studies suggest similar results [163]. There is a high variability in response to HBOT in preclinical and clinical studies, which is likely attributed to insufficient understanding of its mechanisms of action, failure to develop and test standardized protocols of treatment, lack of knowledge regarding its interactions as an adjuvant to standard care, and individual patient and tumor-type responses. Clearly HBOT offers significant potential as a cancer treatment, but we must continue to study its effects in animal models to determine exactly when, where, and how it will be most beneficial clinically. 71

89 2.4.2 Anti-Cancer Effects of Hyperbaric Oxygen Therapy Inhibition of Angiogenesis As described, tumor hypoxia initiates a positive feedback cycle wherein the new, immature blood vessels formed promote further hypoxia and angiogenesis. HBOT saturates tumors with oxygen, which can inhibit angiogenic signaling, limit new blood vessel formation, and prevent further tumor growth. Many of these effects are likely due to inhibition of HIF-1, a potent enhancer of angiogenic activators like VEGF and PDGF. Pigment epithelium derived factor (PEDF), an inhibitor of angiogenesis, is also stimulated by hyperoxia [186]. A small number of animal studies have demonstrated that HBOT can reduce angiogenesis and blood vessel density in vivo. Stuhr et al reported that HBOT and normobaric hyperoxia decreased vascular density and tumor growth in DMBA-induced rat mammary tumors [166, 167]. Proangiogenic genes including VEGFα, VEGFβ, FGF, PDGF, and TGF-α were down-regulated in HBOT-treated tumors. Similarly, HBOT reduced both central and peripheral blood vessel density in rat glioma xenografts [175] Inhibition of HIF-1 Signaling HIF-1 activation is dependent on tissue po2. Its regulatory subunit, HIF-1α, is rapidly degraded under normal oxygen tension [187]. HBOT significantly increases intratumoral po2 [188]. Restoring normal oxygenation can potentially off-set the tumor-promoting effects of HIF- 1 signaling in the tumor. The effects of HBOT on HIF-1α expression in cancer have not been well-investigated. However, a number of studies in focal or global brain ischemia models indicate that HBOT decreases HIF-1α in the striatum, hippocampus, and cortex of rats [189, 72

90 190]. HBOT would likely elicit similar effects in hypoxic tumors. HBOT down-regulated the expression of several HIF-responsive genes in a rat mammary tumor, but the authors did not report its effect on HIF-1α specifically [167]. As the downstream effects of HIF-1 activation are so widely implicated in cancer progression, invasion, and metastasis, it is important to characterize the effects of HBOT on this signaling cascade in neoplastic cells. It is likely, however, that HBOT will inhibit HIF-1 expression in tumors as it does in healthy tissues Enhanced ROS Production Breathing pure oxygen at greater than 1 ATA increases ROS production in the tissues [191]. D Agostino and Dean demonstrated that HBOT elicits significant oxidative stress in U87 glioma cells as shown by enhanced lipid peroxidation and plasma membrane blebbing measured with atomic force microscopy [192]. These effects were reduced with the antioxidant trolox-c, confirming that the lipid peroxidation was ROS-mediated. Membrane blebbing is a consistent feature of apoptosis, suggesting that HBOT sensitized the U87 cells to cell death by the production of ROS [193]. Both intrinsic and extrinsic activation of apoptosis depend on ROS signaling [194]. Fas ligand (Fas-L) increases ROS production which serve as a necessary signal for recruitment of the Fas-associated death domain (FADD) and subsequent activation of the caspase cascade in the extrinsic pathway [195]. Similarly, ROS stabilize the mitochondria permeability transition pore allowing release of cytochrome c, the widely regarded point of no return for induction of the intrinsic apoptotic pathway [196]. It is important to note there appears to be a relative selectivity of pro-oxidant therapies in that levels which are cytotoxic to cancer cells are significantly less so to their normal tissue 73

91 counterparts [196]. We have seen similar results in our laboratory by comparing ROS production in healthy brain cells versus brain tumor cells following HBOT (unpublished data). This may be one consequence of the state of elevated ROS in which cancer cells exist. Comparing basal ROS production between oncogene-transformed and normal cells of the same cell type show a clear increase in ROS production following transformation [197, 198]. Wang and colleagues suggested that while cancers benefit from this elevation in ROS, this pushes them closer to, and thus more susceptible to, surpassing the threshold level which inevitably induces cell death [196]. Thus it is possible to incite cell death with even modest increases in oxidative stress [196]. Mitochondrial damage likely underlies the differential increase in ROS production between cancer and normal cells following pro-oxidant therapies like HBOT [64]. Increasing po2 in tumor cells with faulty mitochondria could further increase the capture of electrons by molecular oxygen, leading to an exacerbated oxidative response Induction of Mesenchymal-to-Epithelial Transition As described, hypoxia and HIF-1 signaling mediate many components of the EMT, including TGF-β, Notch, SNAIL, SLUG, TWIST, and ZEB. HBOT increases intratumoral oxygen tensions, suggesting that it would inhibit HIF-1 activity and consequently its downstream effectors. Moen and colleagues investigated the effects of HBOT on EMT in a rat model of DMBA-induced mammary adenocarcinoma [167]. Tumors were allowed to grow for 5 weeks before treated rats received four 90-minute sessions of HBOT (100% O2, 2 bar) over a 10 day period. While the tumors in control animals continued to grow significantly during the treatment period, tumors in HBOT-treated rats shrunk in size. Tumors were excised and analyzed for 74

92 morphological, vascular, and gene expression changes. HBOT-treated tumors had an increased expression of epithelial markers, including AREG, KRT14, KRT5, CDH1, PERP, DSP, SerpinB, ODN, and CDH3. The mesenchymal markers SNAIL2, FGFR1, CDH2, fibronectin-1, CDH11, S100α4, BMP-7, FZD2, TWIST2, NID1, and vimentin were significantly down-regulated. This study clearly demonstrates the potential for HBOT to reverse and inhibit the EMT phenotype and suggests that it may be effective at preventing or treating metastatic disease Potential Concerns or Side Effects Stimulating Angiogenesis HBOT is used clinically to promote the healing of chronic wounds [199]. Because one of the major mechanisms by which HBOT enhances wound healing is through the stimulation of angiogenic signaling, early studies predicted that it would elicit similar effects in cancer [200]. Interestingly, and for reasons not well-understood, decades of research since have shown that HBOT actually elicits opposite effects in wounds and tumors [201]. Feldmeier and colleagues proposed potential contributing factors for the differential response to HBOT between wounds and tumors: (1) wounds involve negative space while tumors grow in limited physical space, (2) tumors are largely unresponsive to negative feedback signals from nearby tissues while wounds respond appropriately, and (3) tumors form abnormal vasculature with chaotic blood flow while wounds regrow normal, functional blood vessels [201]. Any or all of these characteristics may contribute in part to the poorly understood differential effects of HBOT on angiogenesis in wounds and tumors. 75

93 Soft Tissue Radionecrosis Clinical studies suggest that when administered concurrently with radiation, HBOT can improve response to therapy, but may also enhance soft tissue necrosis in surrounding normal tissue [202]. However, Kohshi and colleagues demonstrated that these adverse effects can be ameliorated by administering HBOT immediately prior to, not concurrent with, radiotherapy [176]. 2.5 VM-M3 Model of Metastatic Cancer Metastasis is responsible for over 90% of cancer-related deaths [203]. Extent of metastatic spread is the best predictor of clinical outcome, with patient prognosis worsening significantly once a primary tumor has spread to distal tissues. For example, while the 5-year survival rate for stage 1 breast cancer is 98%, it drops to 16-20% in metastatic breast cancer patients [204]. Currently, there are very few treatments effective for long-term management of metastatic disease. Radiation and many chemotherapies are often successful at shrinking primary and metastatic tumors initially, but some portion of cancer cells often survive initial treatment. They exist in a dormant state as minimal residual disease for some period of time before re-emerging and re-establishing tumor foci. These recurrent tumors are often very resistant to further treatment and account for the majority of cancer deaths. Perhaps one of the biggest obstacles preventing the development of permanently effective treatments for metastatic disease is the lack of preclinical models which reflect the metastatic behavior of malignant human tumors. Xenograft models involve the implantation of human cancer cells in immunodeficient rodents and are commonly used in preclinical research. However, these tumors 76

94 often do not metastasize, even when created from highly aggressive cancers. The immune system is known to play a critical role in cancer progression and metastasis. Thus, perhaps it is not surprising that many therapies that are effective in xenograft models provide little to no therapeutic efficacy in human clinical trials. Transgene or carcinogen-induced models are also used, but typically only form metastases in a portion of animals. It is also unclear how similar the tumorigenic pathology of these models are to spontaneous tumors. Tail-vein injection models often form metastatic lesions but eliminate some of the most critical aspects of metastatic spread including escape from the confines of the primary tumor and intravasation into the vasculature. The VM-M3 model of metastatic cancer was developed from a spontaneous brain tumor in a mouse of the VM/dk inbred strain by Dr. Thomas Seyfried of Boston College [205]. VM/dk mice have an unusually high incidence (approximately 1.5%) of spontaneous brain tumors [206, 207]. This is approximately a 210-fold increase in incidence compared to C57BL/6J mice [208]. Most of the spontaneous tumors in VM/dk mice are characterized as astrocytomas [206, 207]. VM/dk mouse brains have abnormal mitochondrial phospholipid composition and altered activity of the ETC complexes compared to C57BL/6J mice, characteristics which have been proposed as a potential explanation for their unique propensity for brain tumors [208]. As previously described, abnormalities in mitochondrial lipids and ETC complex function is a very common feature of human cancers. The VM-M3 tumor was adapted to cell culture and transduced with a lentivirus vector containing firefly luciferase under control of the cytomegalovirus promoter allowing for in vivo bioluminescent imaging [205]. VM-M3 cells are highly invasive and metastatic in vivo. Intracerebral implantation induces the formation of a malignant brain tumor that behaves remarkably similar to human glioblastoma multiforme 77

95 (GBM). VM-M3 cells invade both the ipsilateral and contralateral hemispheres, and migrate through the CNS via the Secondary Structures of Scherer, the unique pattern of human GBM invasion described by Hans Scherer in the 1940s [209]. Some animals even exhibit extraneural metastases from orthotopic implantation. It is commonly thought that primary brain tumors cannot metastasize outside the CNS, but this should be reevaluated based on a significant amount of clinical data that suggests otherwise. Indeed, numerous case reports have confirmed secondary metastatic GBM lesions in the peripheral tissues in humans [ ]. One report even described the rapid development of metastatic cancer of GBM origin in multiple transplant recipients who received organs from a patient who died of a primary GBM [220]. A review of the literature clearly demonstrates that primary brain cancer can form systemic metastases if they gain access to extracranial sites. Considering the poor prognosis of malignant brain cancer, it is thus likely that most patients simply die of primary tumor burden prior to extraneural metastasis or before metastatic lesions become symptomatic. Thus, the metastatic behavior of VM-M3 cells represents another characteristic of human disease and further supports its use as a preclinical model of malignant cancer. The remarkable metastatic potential of VM-M3 cells led Seyfried and his team to create the VM-M3 model of metastatic cancer. Subcutaneous implantation of VM-M3 cells results in rapid and systemic metastasis within approximately 2-3 weeks that is highly reproducible [205]. Primary sites of metastasis include liver, lungs, kidney, spleen, adipose, intestine, bone marrow, and brain. In culture, VM-M3 cells exist in a mixed morphology of large flat cells with protoplasmic extensions and small, rounded cells which coordinate with cell cycle progression [205]. The large, flat cells transform into the rounded cells for division, and return to their prior morphology after mitotic completion. VM-M3 cells, like many malignant cells of various cancer 78

96 types, exhibit multiple molecular and behavioral characteristics of mesenchymally-derived macrophages [ ]. As previously described, the remarkable similarities between metastatic cells and macrophages has led some researchers to hypothesize that macrophage transformation or tumor-macrophage fusion in the tumor microenvironment may play a pivotal role in metastasis [221, ]. VM-M3 cells are highly adhesive as well as phagocytotic, as demonstrated by their ability to uptake fluorescent beads in culture. They express high levels of the CXCR4 chemokine receptor, which is considered to be a marker for highly invasive GBM as well as microglia/macrophages [205]. They also express many other cell surface markers of microglia/macrophages, including Iba1, CD11b, F4/80, CD68, and CD45 and have a lipid composition profile closely resembling macrophages as well [205]. VM-M3 cells transformed spontaneously in vivo, and they are grown in immunocompetent VM/dk hosts with their syngeneic genetic background. VM-M3 cells spread naturally from the s.c. adipose, thus replicating all of the major steps of metastasis, from escaping the confines of the site of inoculation to the establishment of metastatic lesions in distal organs. Like most metastatic cancers, but unlike most cancer models, VM-M3 cells are exceptionally aggressive and difficult to kill in vitro and in vivo. These factors likely account for their remarkable molecular and behavioral similarities with human disease and make the VM-M3 model a very good choice for preclinical investigation of novel therapeutics. 2.6 Central Hypothesis and Project Aims The metabolic abnormalities of cancer cells provide a plethora of therapeutic targets that have been largely unrealized to date. As described, there is substantial scientific rationale for 79

97 investigating the anti-cancer efficacy of the KD, ketone supplementation, and HBOT. The metabolic defects targeted by these non-toxic therapies are largely universal between cancer types, and are especially prominent in metastatic lesions. As metastasis is the cause of nearly all cancer-related deaths, it is critically important to test the efficacy of novel therapies on preclinical models which exhibit metastatic behavior. The KD and HBOT have been investigated to a limited extent in primary cancer models, and this preliminary evidence suggests a substantial need for further investigation. Furthermore, the effect of the KD and HBOT on aggressive or metastatic cancer is largely unknown. Ketone supplementation is a novel treatment that may inhibit cancer progression and metastasis in a variety of ways but has not been tested for its therapeutic efficacy. We hypothesize that ketosis and hyperbaric oxygen metabolic therapies will elicit anti-cancer effects in the VM-M3 model of metastatic cancer when administered individually and as a multi-combination treatment. The major goals of this research project were to characterize the effects of ketosis and HBOT on tumor growth, metastatic spread, and survival in VM-M3 mice in vivo, to corroborate their effects on VM-M3 cells in vitro, and to provide preliminary insight into the cellular and molecular effects of these therapies for future mechanistic studies. If effective, these treatments could provide novel, nontoxic adjuvant treatments for many cancer patients, including those with aggressive or late-stage cancers who currently have very few therapeutic options. 2.7 Closing Remarks In this chapter we have provided a review of the literature and scientific rationale for three potential metabolic therapies for cancer: the ketogenic diet, ketone supplementation, and 80

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113 CHAPTER 3: THE KETOGENIC DIET AND HYPERBARIC OXYGEN INHIBIT VM-M3 CANCER IN VITRO AND IN VIVO 3.1 Chapter Synopsis In this chapter we present data demonstrating the effects of the KD and HBOT on VM- M3 cancer in vitro and in vivo. Both therapies elicited a trend of reduced tumor growth and metastatic spread in vivo, but only the KD, not HBOT, prolonged survival when administered as a stand-alone therapy. However, combining the KD with HBOT resulted in a synergistic effect, increasing mean survival time by 78% compared to controls. HBOT treatment decreased HIF-1α in vitro, an effect which likely contributes significantly to its use as a cancer therapy. Portions of this chapter have been previously published in Poff A, Ari C, Seyfried T, D Agostino D. (2013) The Ketogenic Diet and Hyperbaric Oxygen Therapy Prolong Survival in Mice with Systemic Metastatic Cancer. PLOS One and a copy of this article may be found in Appendix C. See Appendix B for copyright permissions. Materials and methods for the studies presented in this chapter can be found in Appendix A. 96

114 3.2 The Ketogenic Diet and Hyperbaric Oxygen Inhibit VM-M3 Cancer in Vitro and in Vivo As described, the KD and HBOT are two potential therapies which can be used to exploit the metabolic deficiencies of cancer cells. In normal tissues, decreased oxygen availability inhibits mitochondrial production of ATP, stimulating an up-regulation of glycolytic enzymes to meet energy needs by substrate level phosphorylation production of ATP. Thus, the cellular response to tumor hypoxia is mediated by several of the same pathways that are overly active in cancer cells with mitochondrial damage and high rates of aerobic glycolysis. This suggests that the ketogenic diet and HBO2T could target several overlapping pathways and tumorigenic behaviors of cancer cells. We hypothesized that these treatments would work individually and synergistically to inhibit tumor progression. We suggest that the addition of these non-toxic adjuvant therapies to the current standard of care may improve progression free survival in patients with advanced metastatic disease. Figure 3.1. The KD and HBOT increases survival time in mice with systemic metastatic cancer. (A) Kaplan-Meier survival plot of study groups. Animals receiving KD and KD+HBOT showed significantly longer survival compared to control animals (p= and p=0.0035, 97

115 respectively; Kaplan-Meier and LogRank Tests for survival distribution). (B) Treatment group cohort size and mean survival times shown. KD mice exhibited a 56.7% increase in mean survival time compared to controls (p=0.0044; two-tailed student s t-test); KD+HBOT mice exhibited a 77.9% increase in mean survival time compared to controls (p=0.0050; two-tailed student s t-test). Results were considered significant when p<0.05. Figure 3.2. Tumor bioluminescence in mice. Tumor growth was slower in mice fed the KD than in mice fed the SD. (A) Representative animals from each treatment group demonstrating tumor bioluminescence at day 21 after tumor cell inoculation. Treated animals showed less bioluminescence than controls with KD+HBOT mice exhibiting a profound decrease in tumor bioluminescence compared to all groups. (B) Total body bioluminescence was measured weekly as a measure of tumor size; error bars represent ±SEM. KD+HBOT mice exhibited significantly less tumor bioluminescence than control animals at week 3 (p=0.0062; two-tailed student s t- test) and an overall trend of notably slower tumor growth than controls and other treated animals throughout the study. (C,D) Day 21 ex vivo organ bioluminescence of SD and KD+HBOT animals (N=8) demonstrated a trend of reduced metastatic tumor burden in animals receiving the 98

116 combined therapy. Spleen bioluminescence was significantly decreased in KD+HBOT mice (*p=0.0266; two-tailed student s t-test). Results were considered significant when p<0.05. Figure 3.3. Effect of KD, HBOT, and KD+HBOT treatment on metastatic spread in VM- M3 mice. In vivo bioluminescent imaging of key metastatic regions in VM-M3 mice 21 days post tumor cell inoculation was performed as an indicator of metastatic tumor size and spread. KD, HBOT, and KD+HBOT treated mice exhibited a trend of reduced spread to the brain, lungs, and liver, but these differences were not significant from controls (p>0.05; One-way ANOVA). Error bars represent ±SEM. 99

117 Figure 3.4. Blood glucose and β-hydroxybutyrate levels in animals. (A) KD-fed mice showed lower blood glucose than controls on day 7 (***p<0.001). Animals in the KD study group had significantly lower blood glucose levels than controls on day 14 (*p<0.05). (B) KD+HBOT mice had significantly higher blood ketones than controls on day 7 (***p<0.001). Error bars represent ±SEM. Blood analysis was performed with One Way ANOVA with Kruksal Wallis Test and Dunn s Multiple Comparison Test post hoc; results were considered significant when p<0.05. Figure 3.5. Animal Weight. Body weight was measured twice a week. Graph indicates average percent of initial body weight animals at days 7 and 14. KD and KD+HBOT mice lost approximately 10% of their body weight by day 7 and exhibited a significant difference in percent body weight change compared to control animals (*p<0.05; ***p<0.001). Error bars represent ±SEM. 100

118 Figure 3.6. Glucose and weight change are correlated to survival. Linear regression analysis revealed a significant correlation between day 7 blood glucose and percent body weight change with survival (p= and p=0.0001, respectively). Results were considered significant when p<

119 Figure 3.7. Effect of KD+HBOT treatment on metastatic liver tumor size and vascularization. Morphology and vascularization of KD+HBOT-treated mouse liver tumors were analyzed by H&E staining and CD31 immunohistochemistry. In general, tumors from KD+HBOT treated mice were smaller with more well-defined borders (A, C) and exhibited a trend of reduced vascularization (B, D, E) that was not significantly different from controls (p>0.05; unpaired t-test). Error bars represent ±SEM. Figure 3.8. HBOT inhibits HIF-1α expression and enhances pakt expression in VM-M3 cells. Expression of cancer and metabolic signaling molecules in VM-M3 cells grown in control media with or without 7 consecutive daily HBOT sessions (100% O2, 2.5 ATA, 90 min) was investigated by western blot analysis. HBOT-treated cells expressed significantly less HIF-1α than cells grown in control media without HBOT (***p<0.001; unpaired t-test). There was a trend of increased pakt expression in HBOT-treated cells, but it was not significantly different from controls (p=0.29; unpaired t-test). There was no effect of treatment on p44 MAPK/Erk1, 102

120 p42 MAPK/Erk 2, or phospho-mtor (data not shown). Error bars represent ±SEM. Results were considered significant when p<0.05. The ketogenic diet and hyperbaric oxygen inhibit VM-M3 cancer in vitro and in vivo Nearly a century after Otto Warburg reported the abnormal energy metabolism of cancer cells, renewed interest in the field has elucidated a plethora of novel therapeutic targets. Two promising treatments involve the use of HBOT to reverse the cancer-promoting effects of tumor hypoxia and the use of the KD to limit the availability of glycolytic substrates to glucoseaddicted cancer cells. Both therapies have been previously reported to possess anti-cancer effects [1-4]. Since these treatments are believed to work by targeting several overlapping mechanisms, we hypothesized that combining these non-toxic treatments would provide a powerful, synergistic anti-cancer effect. Furthermore, since metastasis is responsible for the overwhelming majority of cancer-related deaths, we tested the efficacy of these conjoined therapies on the VM-M3 mouse model of metastatic cancer [5, 6]. We found that the KD fed ad libitum significantly increased mean survival time in mice with metastatic cancer (p=0.0194; Figure 3.1). It is important to note that KD-fed animals lost approximately 10% of their body weight over the course of the study (Figure 3.5). It is well established that low carbohydrate, high fat ketogenic diets can cause body weight loss in overweight humans [7-9]. Ketogenic diets are also known to have an appetite suppressing effect which may contribute to body weight loss [10]. Along with appetite suppression, a potential contributing factor to the observed body weight loss is the possibility that mice found the KD to be less palatable and were self-restricting caloric intake. As calorie restriction is known to elicit 103

121 profound anti-cancer effects, the ketogenic diet may inhibit cancer progression in part by indirect dietary energy restriction [11, 12]. Fine and colleagues recently used a very low carbohydrate KD to promote stable disease or partial remission in patients with advanced metastatic cancer [13]. Fine s study demonstrated a correlation between blood ketones and response to the diet therapy, suggesting that ketone elevation itself also contributes to the anti-cancer efficacy of the KD. HBOT inhibited HIF-1α expression in VM-M3 cells (Figure 3.8). As described, the activity of the HIF-1 transcription factor plays an important role in every aspect of cancer progression, from tumorigenesis to metastasis [14, 15]. Inhibition of HIF-1α, and subsequent inhibition of HIF-1 activity, by HBOT in VM-M3 cells likely plays an important role in its anticancer efficacy both in vitro and in vivo. Although not statistically significant, pakt expression may be slightly enhanced by HBOT. Oxidative stress and HBOT are both known to activate Akt signaling in normal cells [4, 16]. It is likely that HBOT induced oxidative stress in VM-M3 cells is responsible for the elevation in pakt expression observed. Furthermore, in humans, HBOT decreases blood glucose immediately following treatment [17]. There is some evidence to suggest this is caused by improved insulin sensitivity which may be mediated in part by the PI3K/Akt pathway [18] and thus could contribute to the elevation in pakt expression following HBOT in the VM-M3 cells. Although not significant from controls, there was a clear trend of reduced metastatic spread in KD, HBOT, and KD+HBOT treated mice as measured by in vivo bioluminescence imaging of key metastatic regions (Figures 3.2 and 3.3). Tumors from KD+HBOT treated mice were smaller and appeared to have more well-defined borders by morphological analysis, and exhibited a trend of decreased vascularization compared to tumors from control mice (Figure 104

122 3.7). As tumors need to form new blood vessels to grow in size, the decreased vascularization observed in these tumors may play a role in preventing metastatic tumor growth. Additionally, the tumors may be less vascularized because they are smaller than tumors from control mice at this time point. Since HIF-1 signaling stimulates both angiogenesis and tumor growth, it is likely that HBOT-induced inhibition of HIF-1 is contributing to both of these effects in vivo. These observations clearly support that ketosis and HBOT elicit anti-cancer effects in vivo, even when tested in an aggressive model of metastatic cancer. As hypothesized, profound anti-cancer effects were observed in our metastatic mouse model after combining the KD with HBOT. Combining these therapies nearly doubled survival time in mice with metastatic cancer, increasing mean survival time by 24 days compared to control animals (p=0.0050; Figure 3.1). The KD+HBOT-treated mice exhibited significantly decreased bioluminescence compared to controls at week 3 (p=0.0062) and a trend of decreased tumor growth rate throughout the study (Figure 3.2). By day 7, all animals on a ketogenic diet had significantly lower blood glucose levels than controls (Figure 3.4). As it has been shown that tumor growth is directly correlated to blood glucose levels [19], this decrease in blood glucose concentration likely contributed to the trend of decreased tumor bioluminescence and rate of tumor growth seen in KD-fed animals (Figure 3.2). Nebeling et al. demonstrated that the KD significantly decreased glucose uptake in pediatric brain tumor patients by FDG-PET analysis [20]. This clinical data suggests decreased glucose delivery to the tumor is a causal mechanism in KD treatment. All KD-fed mice showed a trend of elevated blood ketones throughout the study; however, only KD+HBOT mice had significantly higher ketones than controls on day 7 (Figure 3.4). As ketones are metabolized exclusively within the mitochondria, cancer cells with damaged mitochondria are unable to adequately use them for energy. Many 105

123 cancers do not express the Succinyl-CoA: 3-ketoacid CoA-Transferase (SCOT) enzyme which is required for ketone body metabolism [21, 22]. In fact, βhb administration prevents healthy hippocampal neurons but not glioma cells from glucose withdrawal-induced cell death [23]. Furthermore, ketone bodies have anti-cancer effects themselves, possibly through inhibition of glycolytic enzymes [24]. Skinner and colleagues demonstrated that acetoacetate and βhb administration inhibits brain cancer cell viability in vitro [25]. Thus, the elevated ketone levels in the KD+HBOT mice likely enhanced the efficacy of this combined therapy. Potential concern may arise regarding the use of a diet therapy for cancer patients susceptible to cachexia. While low carbohydrate or ketogenic diets promote weight loss in overweight individuals, they are also known to spare muscle wasting during conditions of energy restriction and starvation [26-29]. In an animal model of cancer cachexia, administration of a low carbohydrate, high fat diet prevented weight loss of the animals while simultaneously decreasing tumor size [30]. Similar effects were described in human cancer patients [20, 31]. The anti-cachexia effects of the KD are not surprising when considering a metabolic switch to fat metabolism and subsequent ketosis evolved as a method of sparing protein during prolonged fasting or starvation [26, 32, 33]. It makes sense that dietary-induced therapeutic ketosis in a cancer patient would prevent muscle wasting similarly as it does with athletes undergoing intense exercise [34]. Furthermore, when given as an adjuvant treatment to advanced cancer patients, the KD improves quality of life and enhances the efficacy of chemotherapy treatment in the clinic [35, 36]. This and other emerging evidence calls into question the common medical advice of limiting fat consumption in overweight cancer patients [37]. Veech and colleagues described the mechanisms by which ketone metabolism protects cells from oxidative damage [28, 32], while more recent evidence suggests that ketones function 106

124 as HDAC inhibitors [38]. βhb metabolism results in an increased reduction of the NAD couple and increased oxidation of the co-enzyme Q inside the mitochondria. Increased oxidation of Q decreases semiquinone levels, subsequently decreasing superoxide anion production [28]. Increased reduction of the NADP couple enhances regeneration of reduced glutathione, an important endogenous antioxidant [28]. Thus, ketone body metabolism protects cells from oxidative damage by decreasing ROS production and by enhancing endogenous antioxidant capabilities. As previously discussed, cancer cells are unable to effectively metabolize ketone bodies; therefore, we do not expect that ketones would confer the same protective effects onto the cancer cell. HBOT increases ROS production within the cell which can lead to membrane lipid peroxidation and cell death [39]. Cancer cells with mitochondrial damage and chaotic perfusion naturally produce chronically elevated levels of ROS but are susceptible to oxidative damage-induced cell death with even modest increases in ROS [4, 40]. We propose a potential mechanism of KD+HBOT efficacy: the KD weakens cancer cells by glucose restriction and the inherent anti-cancer effects of ketone bodies while simultaneously conferring a protective advantage to the healthy tissue capable of ketone metabolism. This metabolic targeting sensitizes the cancer cells to HBOT-induced ROS production and oxidative damage, contributing to the efficacy of combining KD with HBOT. Additionally, ketone metabolism by the healthy tissues likely confers protection against the potential negative consequences of HBOT (CNS oxygen toxicity) [41-43]. Recent in vivo studies support the neuroprotective effects of ketone esters [44, 45]. These hypothetical mechanisms may contribute to the safety and efficacy of the KD+HBOT combined therapy. Stuhr and Moen recently published a comprehensive review of the literature on the use of HBOT for cancer [46]. The authors concluded that HBOT should be considered a safe treatment 107

125 for patients with varying malignancies and that there is no convincing evidence its use promotes cancer progression or recurrence. In the literature, there are a substantial number of studies indicating that HBOT can induce marked anti-cancer effects in vitro and in animal and human studies alike [4, 46, 47]. Evidence is mixed, however, as other studies reported no effect with HBOT [4, 46]. Indeed, in our present study, HBOT alone did not improve the outcome of VM mice with metastatic cancer, but combining HBOT with KD elicited a dramatic therapeutic effect. Perhaps adding the KD or another metabolic therapy (e.g. 2-deoxyglucose, 3- bromopyruvate) to HBOT would produce similar results in these previously reported studies demonstrating no efficacy due to HBOT alone. It is important to look for synergistic interactions between therapies which may increase the efficacy of cancer treatment. Scheck and coworkers reported complete remission without recurrence in 9 of 11 mice with glioma by combining the KD with radiation [48]. Marsh, et al. reported synergy between the restricted ketogenic diet and the glycolysis inhibitor 2-deoxyglucose [49]. Might adding HBOT to these combination therapies elicit even better results? Similarly, might the use of adjuvant therapies like KD and HBOT enhance patient response to standard care? Our study strongly suggests that combining a KD with HBOT may be an effective nontoxic therapy for the treatment of metastatic cancer. The efficacy of combining these non-toxic treatments should be further studied to determine their potential for clinical use. Based on the reported evidence, it is highly likely that these therapies would not only contribute to cancer treatment on their own, but might also enhance the efficacy of current standard of care and improve the outcome of patients with metastatic disease. 108

126 3.3 References for Chapter 3 1. Stuhr L, Iversen V, Straume O, Maehle B, Reed R: Hyperbaric oxygen alone or combined with 5-FU attenuates growth of DMBA-induced rat mammary tumors. Cancer letters 2004, 210: Scheck A, Abdelwahab M, Fenton K, Stafford P: The ketogenic diet for the treatment of glioma: Insights from genetic profiling. Epilepsy Res 2011, 100: Seyfried T, Shelton L: Cancer as a metabolic disease. Nutr Metab (Lond) 2010, 7(2cd e-d9e1-b540-9c7fa45dcb27):7. 4. Daruwalla J, Christophi C: Hyperbaric oxygen therapy for malignancy: a review. World journal of surgery 2006, 30: Shelton L, Mukherjee P, Huysentruyt L, Urits I, Rosenberg J, Seyfried T: A novel preclinical in vivo mouse model for malignant brain tumor growth and invasion. Journal of neuro-oncology 2010, 99(4d2fef ae9a-075c-9c7fa45feed8): Huysentruyt LC, Mukherjee P, Banerjee D, Shelton LM, Seyfried TN: Metastatic cancer cells with macrophage properties: evidence from a new murine tumor model. International journal of cancer 2008, 123(1): Astrup A, Ryan L, Grunwald G, Storgaard M, Saris W, Melanson E, Hill J: The role of dietary fat in body fatness: evidence from a preliminary meta-analysis of ad libitum low-fat dietary intervention studies. The British journal of nutrition 2000, 83:S Volek J, Sharman M, Gómez A, Judelson DA, Rubin M, Watson G, Sokmen B, Silvestre R, French D, Kraemer W: Comparison of energy-restricted very low-carbohydrate and low-fat diets on weight loss and body composition in overweight men and women. Nutr Metab (Lond) 2004, 1(1): Hussain TA, Mathew TC, Dashti AA, Asfar S, Al-Zaid N, Dashti HM: Effect of lowcalorie versus low-carbohydrate ketogenic diet in type 2 diabetes. Nutrition 2012, 28(10): Johnstone A, Horgan G, Murison S, Bremner D, Lobley G: Effects of a high-protein ketogenic diet on hunger, appetite, and weight loss in obese men feeding ad libitum. The American journal of clinical nutrition 2008, 87(1): Hursting S, Smith S, Lashinger L, Harvey A, Perkins S: Calories and carcinogenesis: lessons learned from 30 years of calorie restriction research. Carcinogenesis 2010, 31(1): Seyfried TN: Cancer as a Metabolic Disease: On the Origin, Management, and Prevention of Cancer. Hoboken, NJ: John Wiley & Sons, In.; Fine E, Segal-Isaacson C, Feinman R, Herszkopf S, Romano M, Tomuta N, Bontempo A, Negassa A, Sparano J: Targeting insulin inhibition as a metabolic therapy in advanced cancer: a pilot safety and feasibility dietary trial in 10 patients. Nutrition 2012, 28(10): Mabjeesh NJ, Amir S: Hypoxia-inducible factor (HIF) in human tumorigenesis. Histol Histopathol 2007, 22(5): Jiang J, Tang Y-l, Liang X-h: EMT: a new vision of hypoxia promoting cancer progression. Cancer Biol Ther 2011, 11(8): Wang X, McCullough KD, Franke TF, Holbrook NJ: Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival. J Biol Chem 2000, 275(19):

127 17. Peleg RK, Fishlev G, Bechor Y, Bergan J, Friedman M, Koren S, Tirosh A, Efrati S: Effects of hyperbaric oxygen on blood glucose levels in patients with diabetes mellitus, stroke or traumatic brain injury and healthy volunteers: a prospective, crossover, controlled trial. Diving Hyperb Med 2013, 43(4): Wilkinson D, Chapman IM, Heilbronn LK: Hyperbaric oxygen therapy improves peripheral insulin sensitivity in humans. Diabetic medicine : a journal of the British Diabetic Association 2012, 29(8): Seyfried T, Sanderson T, El-Abbadi M, McGowan R, Mukherjee P: Role of glucose and ketone bodies in the metabolic control of experimental brain cancer. Br J Cancer 2003, 89(62175f06-e e22-9c7fa647593a): Nebeling LC, Miraldi F, Shurin SB, Lerner E: Effects of a ketogenic diet on tumor metabolism and nutritional status in pediatric oncology patients: two case reports. Journal of the American College of Nutrition 1995, 14(2): Tisdale M, Brennan R: Loss of acetoacetate coenzyme A transferase activity in tumours of peripheral tissues. Br J Cancer 1983, 47(2): Sawai M, Yashiro M, Nishiguchi Y, Ohira M, Hirakawa K: Growth-inhibitory effects of the ketone body, monoacetoacetin, on human gastric cancer cells with succinyl- CoA: 3-oxoacid CoA-transferase (SCOT) deficiency. Anticancer research 2004, 24(4): Maurer G, Brucker D, Bähr O, Harter P, Hattingen E, Walenta S, Mueller-Klieser W, Steinbach J, Rieger J: Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy. BMC Cancer 2011, 11: Magee BA, Potezny N, Rofe AM, Conyers RA: The inhibition of malignant cell growth by ketone bodies. Aust J Exp Biol Med Sci 1979, 57(5): Skinner R, Trujillo A, Ma X, Beierle E: Ketone bodies inhibit the viability of human neuroblastoma cells. Journal of pediatric surgery 2009, 44(1): Manninen AH: Very-low-carbohydrate diets and preservation of muscle mass. Nutr Metab (Lond) 2006, 3: Cahill G: Fuel metabolism in starvation. Annual review of nutrition 2006, 26: Veech RL: The therapeutic implications of ketone bodies: the effects of ketone bodies in pathological conditions: ketosis, ketogenic diet, redox states, insulin resistance, and mitochondrial metabolism. Prostaglandins, leukotrienes, and essential fatty acids 2004, 70(3): Volek J, Sharman M, Love D, Avery N, Gómez A, Scheett T, Kraemer W: Body composition and hormonal responses to a carbohydrate-restricted diet. Metabolism: clinical and experimental 2002, 51(7): Tisdale MJ, Brennan RA, Fearon KC: Reduction of weight loss and tumour size in a cachexia model by a high fat diet. Br J Cancer 1987, 56(1): Nebeling LC, Lerner E: Implementing a ketogenic diet based on medium-chain triglyceride oil in pediatric patients with cancer. J Am Diet Assoc 1995, 95(6): Veech RL, Chance B, Kashiwaya Y, Lardy HA, Cahill GF, Jr.: Ketone bodies, potential therapeutic uses. IUBMB Life 2001, 51(4):

128 33. Wu GY, Thompson JR: The effect of ketone bodies on alanine and glutamine metabolism in isolated skeletal muscle from the fasted chick. The Biochemical journal 1988, 255(1): Paoli A, Grimaldi K, D'Agostino D, Cenci L, Moro T, Bianco A, Palma A: Ketogenic diet does not affect strength performance in elite artistic gymnasts. Journal of the International Society of Sports Nutrition 2012, 9(1): Stafford P, Abdelwahab M, Kim DY, Preul M, Rho J, Scheck A: The ketogenic diet reverses gene expression patterns and reduces reactive oxygen species levels when used as an adjuvant therapy for glioma. Nutr Metab (Lond) 2010, 7: Schmidt M, Pfetzer N, Schwab M, Strauss I, Kämmerer U: Effects of a ketogenic diet on the quality of life in 16 patients with advanced cancer: A pilot trial. Nutr Metab (Lond) 2011, 8(1): Champ C, Volek J, Siglin J, Jin L, Simone N: Weight gain, metabolic syndrome, and breast cancer recurrence: are dietary recommendations supported by the data? International journal of breast cancer 2012, 2012: Shimazu T, Hirschey MD, Newman J, He W, Shirakawa K, Le Moan N, Grueter CA, Lim H, Saunders LR, Stevens RD et al: Suppression of oxidative stress by betahydroxybutyrate, an endogenous histone deacetylase inhibitor. Science 2013, 339(6116): D'Agostino D, Olson J, Dean J: Acute hyperoxia increases lipid peroxidation and induces plasma membrane blebbing in human U87 glioblastoma cells. Neuroscience 2009, 159: Aykin-Burns N, Ahmad I, Zhu Y, Oberley L, Spitz D: Increased levels of superoxide and H2O2 mediate the differential susceptibility of cancer cells versus normal cells to glucose deprivation. The Biochemical journal 2009, 418: D'Agostino D, Pilla R, Held H, Landon CS, Ari C, Arnold P, Dean JB: Development, testing, and therapeutic applications of ketone esters (KE) for CNS oxygen toxicity (CNS-OT); i.e., hyperbaric oxygen (HBO2)-induced seizures. In: FASEB. San Diego, CA; Pilla R, D'Agostino D, Landon C, Dean J: Intragastric ketone esters administration prevents central nervous system oxygen toxicity via tidal volume and respiratory frequency modulation in rats. In: Third International Symposium on Dietary Therapies for Epilepsy & Other Neurological Disorders Bennett A, Ari C, Kesl S, Luke J, Diamond D, Dean JB, D'Agostino DP: Effect of ketone treatment and glycolysis inhibition in brain cancer cells (U87MG) and rat primary cultured neurons exposed to hyperbaric oxygen and amyloid beta. In: FASEB J D'Agostino DP, Pilla R, Held HE, Landon CS, Puchowicz M, Brunengraber H, Ari C, Arnold P, Dean JB: Therapeutic ketosis with ketone ester delays central nervous system oxygen toxicity seizures in rats. Am J Physiol Regul Integr Comp Physiol Kashiwaya Y, Bergman C, Lee JH, Wan R, King MT, Mughal MR, Okun E, Clarke K, Mattson MP, Veech RL: A ketone ester diet exhibits anxiolytic and cognition-sparing properties, and lessens amyloid and tau pathologies in a mouse model of Alzheimer's disease. Neurobiol Aging 2013, 34(6): Moen I, Stuhr LE: Hyperbaric oxygen therapy and cancer-a review. Targeted oncology 2012, 7:

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130 CHAPTER 4: KETONE SUPPLEMENTATION INHIBITS VM-M3 CANCER IN VITRO AND IN VIVO 4.1 Chapter Synopsis Here we describe the effects of ketone supplementation on VM-M3 cancer in vitro and in vivo. When administered with both standard and ketogenic diets, ketone supplementation decreased tumor growth and metastatic spread and extended mean survival time in VM-M3 mice. βhb supplementation inhibited VM-M3 cell proliferation and viability in vitro, but had no effect on ROS production. It also inhibited expression of HIF-1α and pakt, mechanisms which are known to elicit anti-cancer effects in vivo. We also investigated the effects of chronic ketone administration on healthy rats, and found that it lowered blood glucose, elevated blood ketones, did not affect blood triglycerides or cholesterol, but did alter the concentration of numerous blood metabolites which have implications for its anti-cancer effects. Portions of this chapter have been previously published in Poff A, Ari C, Arnold P, Seyfried T, D Agostino D. (2014) Ketone Supplementation Decreases Tumor Cell Viability and Prolongs Survival of Mice with Metastatic Cancer. Int. J. of Cancer. and a copy of this article may be found in Appendix C. See Appendix B for copyright permissions. Materials and methods for the studies presented in this chapter can be found in Appendix A. 113

131 4.2 Ketone Supplementation Inhibits VM-M3 Cancer in Vitro and in Vivo Independently of Glucose Concentration While the literature strongly suggests that ketone bodies impede cancer growth in vitro, the in vivo efficacy of dietary ketone supplementation has not been adequately examined. It is possible to raise blood ketone levels without the need for carbohydrate restriction by administering a source of supplemental ketones or ketone precursors. 1,3-butanediol (BD) is a commercially available food additive and hypoglycemic agent that is converted to βhb by the liver [32, 33]. The ketone ester (KE) elevates both AcAc and βhb in a dose-dependent manner to levels beyond what can be achieved with the KD or therapeutic fasting [34]. Oral administrations of BD and KE have been shown to elevate blood ketones for at least 240 min in rats [34]. Since ketone bodies appear to elicit anti-cancer effects, and metastasis is the most significant obstacle in the successful treatment of neoplasms, we tested the efficacy of ketone supplementation the VM-M3 cell line and mouse model of metastatic cancer. 114

132 Figure 4.1. βhb decreases VM-M3 cell proliferation and viability in vitro. (A) VM-M3 proliferation was inhibited when grown in low (3mM) glucose and/or ketone supplemented (5mM βhb) media (Glu: glucose; βhb: β-hydroxybutyrate). Cell density was significantly less in low (3mM) glucose conditions and in both low (3mM) and high (25mM) glucose conditions with ketone supplementation (5mM βhb ) at 24, 48, 72, 96, 120, and 144 hours compared to high glucose controls (p<0.05; One-Way ANOVA). Although low (3mM) glucose with ketone supplementation (5mM βhb) demonstrated a trend of decreased proliferation compared to low (3mM) glucose alone, the difference was not statistically significant. (B) VM-M3 viability was decreased when grown in low (3mM) glucose conditions and in both low (3mM) and high (25mM) glucose conditions with ketone supplementation (5mM βhb). There was a significantly smaller percentage of live cells in all treated versus control (high glucose, 25mM) media (*p<0.05, ***p<0.001; Unpaired t-test). (C-F) Live/dead fluorescence of VM-M3 cells grown in high (25mM) glucose with and without 5mM βhb. Results were considered significant when p<0.05. Error bars represent ±SEM. 115

133 Figure 4.2. βhb inhibits HIF-1α and pakt expression in VM-M3 cells. Expression of cancer and metabolic signaling molecules in VM-M3 cells grown for 7 days in control media with or without 5mM βhb was investigated by western blot analysis. βhb-treated cells expressed significantly less HIF-1α and pakt than cells grown in control media without βhb (*p<0.05, ***p<0.001; unpaired t-test). There was no effect of treatment on p44 MAPK/Erk1, p42 MAPK/Erk 2, or phospho-mtor (data not shown). Error bars represent ±SEM. Results were considered significant when p<

134 Figure 4.3. Effect of supplemental ketones on tumor bioluminescence. Ketone supplement fed mice demonstrated a trend of slower tumor growth and metastatic spread compared to controls. (A) Representative animals from each group at 21 days post tumor cell inoculation showing whole body tumor bioluminescence. Treated mice exhibited reduced tumor burden and metastatic spread. (B) Whole animal bioluminescence tracked over time as a measure of tumor growth rate. The number of animals alive at each week is presented as the color-coded number above the average bioluminescence data points. (C-E) Bioluminescence of key metastatic regions: brain, lungs, and liver. Treated animals exhibited a trend of slower tumor growth rate and decreased metastatic spread compared to controls, although bioluminescence was not significantly different from controls. Error bars represent ±SEM. Figure 4.4. Supplemental ketones extend survival time in mice with systemic metastatic cancer. (A) Kaplan-Meier survival curve of treatment groups. BD and KE, but not CR, treated mice demonstrated prolonged survival compared to controls (Logrank test for survival distribution; p=0.01, p=0.001, and p=0.30 respectively). (B) Cohort size, mean survival, and percent change from mean survival of controls shown. BD mice exhibited a 51% increase in mean survival time compared to controls, while KE mice exhibited a 69% increase in mean 117

135 survival time compared to controls (*p<0.05; ***p<0.001; One-Way ANOVA). Results were considered significant when p<0.05. Figure 4.5. Effect of ketone supplementation on blood glucose, blood βhb, urine AcAc, and body weight in mice. (A-C) Glucose and ketones of healthy VM/Dk mice 0-12 hours after feeding following 8 hour fast. BD and KE treated mice demonstrated decreased blood glucose and elevated blood βhb following feeding (*p<0.01; **p<0.05; ***p<0.001; Two-Way ANOVA). KE, but not BD, treated mice demonstrated elevated urine AcAc 12 hours after feeding (***p<0.001; One-Way ANOVA). (D-F) Blood glucose, ketones, and body weight of survival study VM-M3 mice prior to significant disease onset. (D) CR and KE treated mice had lower glucose than controls by day 7 (**p<0.01; ***p<0.001; One-Way ANOVA). (E) CR and 118

136 KE mice had elevated blood βhb compared to controls at day 7 (*p<0.05; ***p<0.001; One- Way ANOVA). (F) CR and KE mice had a 20% reduction in body weight compared to controls at day 14 which was sustained for the duration of study (***p<0.001; One-Way ANOVA). Results were considered significant when p<0.05. Error bars represent ±SEM. (G,H) Blood glucose and ketones of VM-M3 cancer mice over the course of the survival study. Figure 4.6. Decreased blood glucose and weight loss correlated with longer survival. Linear regression analysis of day 7 blood glucose and percent body weight change for all study animals revealed a significant correlation to survival time (p= and p=0.0046, respectively). Results were considered significant when p<0.05. Ketone supplementation inhibits VM-M3 cancer in vitro and in vivo independent of glucose concentration The Warburg Effect is the most ubiquitous cancer phenotype, exhibited by most if not all cancer types [1]. Exploiting the metabolic deficiencies of cancer cells should be prioritized, since this therapeutic strategy would likely prove effective against most cancers. Mitochondrial 119

137 dysfunction underlies many aspects of cancer metabolic deficiency and prevents cancer cells from effectively utilizing ketone bodies for energy [2, 3]. In the current study, ketone supplementation decreased VM-M3 cell proliferation and viability (Figure 4.1), confirming similar results demonstrated in other cancer types in vitro [2, 4, 5]. As described in Chapter 1, HIF-1 is a transcription factor that mediates the cellular response to oxygen deprivation. It is known to be overly active in cancers and plays a major role in all of the hallmarks of cancer including tumorigenesis, proliferation, survival, invasion, and metastasis, and its expression is correlated with poor clinical prognosis [6-8]. Many chemotherapies work in part by inhibiting HIF-1 signaling [9]. Akt is a serine/threonine kinase that mediates numerous signaling cascades including Ras, PI3K, mtor, insulin receptor, as well as other growth factor and cytokine pathways. The kinase activity of Akt is activated by a phosphorylation event on a carboxy terminal serine residue (Ser473). Akt signaling is enhanced in most cancers by multiple mechanisms [10]. In general, its activation elicits a mitogenic effect, enhancing protein synthesis, cell cycle progression, proliferation, and survival. It also enhances the Warburg effect by increasing glucose transport and the activity of the glycolytic enzymes. βhb inhibited both HIF-1α and pakt expression in VM-M3 cells (Figure 4.2). We therefore hypothesized that dietary administration of ketone body precursors would inhibit disease progression in vivo. Indeed, dietary administration of ketone precursors, BD and KE, increased mean survival time by 51% and 69%, respectively, in VM-M3 mice with metastatic cancer (Figure 4.4). These data support the use of supplemental ketone administration as a feasible and efficacious cancer therapy which should be further investigated in additional animal models in order to determine potential for clinical use. 120

138 Ketone supplementation decreased blood glucose following acute administration, decreased body weight with chronic administration, and sustained ketosis in vivo, even when administered with a high carbohydrate rodent chow in both healthy (VM/Dk) and cancer (VM- M3) mice (Figure 4.5). This study demonstrates the ability of dietary administration of BD and KE to significantly elevate ketone bodies in vivo for at least 12 hours in healthy VM/Dk mice and 7 days in VM-M3 cancer mice (Figure 4.5). VM-M3 cancer is very aggressive, and health of control animals declines rapidly after tumor cell inoculation. With onset of disease progression, animal feeding behavior, body weight, and blood metabolites vary considerably. Blood and body weight data prior to the onset of significant disease progression in all treatment groups (Figure 4.5 D-F) demonstrate the effects of the dietary therapies on blood metabolites in VM-M3 cancer mice largely aside from the confounding effects of disease progression. Figure 4.5 G and H show blood glucose and βhb levels of VM-M3 mice over the course of the survival study. Disease progression altered feeding behavior, and animals often exhibited low blood glucose and elevated blood ketones prior to euthanasia. For example, nearly all CR mice were euthanized in the 5 th week of the study, with their declining health reflected by the marked increase in blood ketones at week 4 (Figure 4.5 H). It is important to note that the metabolic changes associated with acute and chronic ketosis are vast and can dramatically affect blood metabolite concentrations. In previous studies, chronic BD and βhb administration have been shown to decrease food intake in the rat and pigmy goat [8, 9]. Similarly, Veech and colleagues demonstrated that feeding a ketone ester supplemented diet increased malonyl-coa, an anorexigenic metabolite known to decrease food intake [11]. Ketone-induced appetite suppression may account for the decreased blood glucose and body weight seen in treated VM-M3 cancer mice (Figure 4.5 F). Additionally, prior studies 121

139 suggest that ketones increase insulin sensitivity [11, 12], which may be contributing to the decreased circulating blood glucose in KE fed mice. Blood metabolite levels are dynamic and change rapidly with food consumption and tissue utilization. Because the VM-M3 cancer mice were fed ad libitum, blood glucose and ketone levels observed at the time of measurement during chronic supplementation were likely affected by variable feeding behavior prior to blood measurement. As mentioned, health status of the animals also altered feeding behavior. Mice reduced food intake and likely experienced cancer-associated cachexia with disease progression, conditions which elevate blood ketones endogenously through beta-oxidation of stored fats. Furthermore, chronic ketosis enhances ketone utilization by tissues, known as keto-adaptation, resulting in lower blood ketone concentrations [13]. All of these factors likely contributed to the observed variability in blood glucose and βhb between acute versus chronic administration of diet and healthy VM/Dk versus VM-M3 cancer mice (Figure 4.5). Previously, dietary intervention in cancer has largely focused on carbohydrate or calorie restriction to exploit the Warburg Effect. CR has been shown to slow disease progression in a variety of cancer models [14-18]. As described, dietary induced ketosis often leads to reduced appetite, decreased calorie consumption, and decreased body weight, creating the possibility that the effects of ketone supplementation could be indirectly due to CR [3]. Interestingly, although CR decreased blood glucose and elevated blood ketones, CR mice exhibited a trend of increased latency to disease progression and increased survival that was not statistically significant from controls in this study (Figures 4.4, 4.5). As described, some data suggest that elevated ketones are responsible for much of the anti-cancer efficacy of the ketogenic diet [19]. Perhaps elevating ketones with exogenous sources such as ketone supplementation or a ketogenic diet, rather than elevating ketones endogenously through lipolysis such as occurs with CR, provides a more 122

140 effective anti-cancer strategy. Additionally, ketone supplementation may preserve lean muscle mass to a greater degree than CR, and may therefore support overall health of the organism in this way. Since CR mice demonstrated a trend of increased latency to disease progression and survival time, and body weight change was correlated to survival (Figure 4.6), indirect CR likely contributed to the efficacy of ketone supplementation but does not account for the significant increase in survival time in ketone-fed mice. Additionally, while BD increased mean survival time by 51%, blood glucose was not significantly lower than in control animals, suggesting that decreased glucose availability was not the primary mechanism of efficacy (Figures 4.4, 4.5). KE treatment increased mean survival to a greater extent than BD (69% increase from control; Figure 4.4). Notably, blood glucose and body weight was more decreased and ketones were more elevated in KE treated animals, possibly due to indirect CR, and perhaps accounts for the enhanced efficacy of KE compared to BD treatment (Figure 4.5). In vitro, 5 mm βhb supplementation decreased proliferation rate and viability of VM-M3 cells, even when added to 25 mm glucose media (Figure 4.1). Indeed, ketone supplementation was as effective as glucoserestriction (low, 3mM glu) treatment in decreasing VM-M3 cell proliferation and viability (Figure 4.1). These data support the in vitro and in vivo conclusions of Fine and others suggesting that ketone bodies can inhibit cancer progression independently of other factors such as carbohydrate or calorie restriction [2, 4, 5, 19]. Listanti and colleagues have published reports that tumor-associated fibroblasts produce ketone bodies to fuel nearby cancer cells [20, 21]. In these reports, the authors co-culture immortalized fibroblasts genetically altered to overexpress rate-limiting ketone production enzymes with breast cancer cells genetically altered to overexpress ketone utilization enzymes. While this phenomenon may occur in the system created by the authors, there is no evidence that 123

141 this situation would occur naturally in vivo. As described, the literature as a whole strongly suggests that cancer cells of various tissue types are unable to effectively use ketone bodies for fuel. Many cancers do not express the succinyl CoA 3-ketoacid CoA transferase (SCOT) enzyme which is necessary for ketone body utilization [22], while several reports have demonstrated a notable deficiency of cancer cell ketone metabolism in vitro [2, 23]. It is also widely accepted that ketone production occurs nearly exclusively in the liver, and there is no known metabolic pathway by which fibroblasts could produce ketones from glucose. Without evidence to support this phenomenon in a natural cellular environment, we continue to accept the notion that most cancer cells cannot effectively metabolize ketones for energy. The ketogenic diet has been shown to enhance the efficacy of both radiation and chemotherapy in vivo [24-26]. As supplemental ketones mimic the physiological ketosis induced by the ketogenic diet, combining supplemental ketone therapy with standard of care could produce similar effects, even if administered with a standard diet. Furthermore, the neuroprotective effects of ketone metabolism have been widely documented [27]. Ketone metabolism protects normal cells from oxidative damage by decreasing mitochondrial ROS production and enhancing endogenous antioxidant defenses [28]. Radiation and chemotherapy work in large part by inducing ROS production in the tumor, but simultaneously incur damage to normal tissue. Ketone metabolism by healthy tissue would likely mitigate some of the adverse side effects of standard of care as ketones have been shown to protect against oxidative stress [28, 29]. Our data strongly suggest that supplemental ketone administration could provide a safe, feasible, and cost-effective adjuvant to standard care that should be further investigated in pre-clinical and clinical settings. 124

142 4.3 Effect of Ketone-Supplemented Ketogenic Diets on VM-M3 Cancer Figure 4.7. Blood glucose, βhb, and body weight of KD+BD and KD+KE treated VM-M3 mice prior to significant disease onset. VM-M3 mice with metastatic cancer were fed either standard rodent chow (control), BD-supplemented ketogenic diet (KD+BD), or KEsupplemented ketogenic diet (KD+KE). (A, C) KD+BD and KD+KE treated mice had significantly decreased blood glucose and body weight compared to control mice by day 7 (**p<0.01, ***p<0.001; one-way ANOVA). (B) KD+BD and KD+KE treated mice had significantly increased blood βhb compared to control mice by day 7 (*p<0.05, ***p<0.001; one-way ANOVA). Error bars represent ±SEM. Results were considered significant when p<

143 Figure 4.8. Effect of KD+BD and KD+KE treatment on tumor growth in VM-M3 mice. VM-M3 mice with metastatic cancer were fed either standard rodent chow (control), BDsupplemented ketogenic diet (KD+BD), or KE-supplemented ketogenic diet (KD+KE). (A) Treated mice exhibited a trend of reduced bioluminescence suggesting lessened tumor burden and metastatic spread compared to controls. (B) Whole animal bioluminescent signal was recorded over time as a measure of tumor growth rate. Treated mice exhibited a trend of slower tumor growth; however, bioluminescent signal was not significantly different compared to controls (p>0.05; one-way ANOVA). 126

144 Figure 4.9. Effect of KD+BD and KD+KE treatment on metastatic spread in VM-M3 mice by in vivo bioluminescent imaging. In vivo bioluminescent imaging of key metastatic regions in VM-M3 mice 21 days post inoculation was performed as an indicator of metastatic tumor size and spread. KD+BD-treated mice exhibited a trend of reduced spread to the brain, and KD+KEtreated mice exhibited a trend of reduced spread to the brain and lungs, but these difference were not significant from controls (p>0.05; One-way ANOVA). Error bars represent ±SEM. 127

145 Figure Effect of KD+KE treatment on metastatic spread in VM-M3 mice by ex vivo bioluminescent imaging. Ex vivo bioluminescent imaging of organs harvested from control and KD+KE treated mice 21 days post inoculation was performed as a more sensitive method of measuring metastatic tumor size and spread. KD+KE-treated mice exhibited a trend of reduced spread to the brain, lungs, kidneys, spleen, adipose, and liver, but this difference was only significant from controls for the adipose tissue (p<0.05; unpaired t-test). Error bars represent ±SEM. Figure Effect of KD+KE treatment on metastatic liver tumor size and vascularization. Morphology and vascularization of KD+KE-treated mouse liver tumors 21 days after VM-M3 cell inoculation were analyzed by H&E staining and CD31 immunohistochemistry. In general, tumors from KD+KE-treated mice were smaller with more well-defined borders (A, C) and exhibited a trend of reduced vascularization (B, D, E) that was not significantly different from controls (p>0.05; unpaired t-test). Error bars represent ±SEM. 128

146 Figure Ketone supplemented ketogenic diets extend survival time in VM-M3 mice with metastatic cancer. VM-M3 mice with metastatic cancer were fed either standard rodent chow (control), BD-supplemented ketogenic diet (KD+BD), or KE-supplemented ketogenic diet (KD+KE). (A) Kaplan-Meier survival curve of study groups. KD+BD and KD+KE treated mice demonstrated significantly prolonged survival compared to controls (p<0.05; log-rank Mantel-Cox test for survival distribution). (B) Cohort size (N), mean survival time, and percent increase in mean survival time from controls shown. KD+BD and KD+KE treated mice exhibited a 61.2% and 65.4% increase in mean survival time compared to controls, respectively (**p<0.01; one-way ANOVA). Results were considered significant when p<0.05. Ketone-Supplemented Ketogenic Diets Prolong Survival in VM-M3 Mice with Metastatic Cancer Please note that the KD+KE survival study data is also included in the manuscript under revision in Chapter 5. It has been included here along with the unpublished BD-supplemented KD data for clarity purposes. Blood glucose and body weight were significantly decreased, and blood βhb significantly increased, in mice receiving ketone supplements prior to significant disease onset (Figure 4.7). These effects were more pronounced in KD+KE treated mice whose body weight loss suggests some degree of calorie restriction (CR). Our study one ketone 129

147 supplementation and body weight matched calorie restricted VM-M3 mice demonstrated that the anti-cancer effects of ketone supplementation may be enhanced by, but is not due to CR alone [30]. Bioluminescent imaging of mice demonstrated a trend of decreased tumor growth rate, but overall tumor burden was not significantly less than controls in either treatment group at 3 weeks (Figure 4.8). A similar result was observed with in vivo bioluminescence of metastatic regions. At 3 weeks, KD+BD treatment elicited a trend of reduced spread to the brain, and KD+KE treatment elicited a trend of reduced spread to the brain and lungs by in vivo bioluminescent imaging; however, these differences were not significantly different from control mice (Figure 4.9). Ex vivo bioluminescent imaging of organs harvested from KD+KE treated mice 21 days post VM-M3 inoculation showed similar results: there was a trend of decreased metastasis to the brain, lungs, liver, spleen, and kidneys, and a significant decrease in metastatic tumor burden in the adipose sample compared to controls (Figure 4.10). Morphological and immunohistochemical analysis of liver tissue demonstrated that KD+KE treated mice had smaller metastatic tumors with more well-defined borders and a trend of decreased vascularity compared to those from control animals, although this difference was not statistically significant (Figure 4.11). The trend of decreased vascularization may be due to an inhibition of angiogenesis, perhaps subsequent to inhibition of HIF-1, a potent stimulator of VEGF expression. Alternatively, inhibition of tumor growth may have precluded the need for angiogenesis at the observed time point in treated mice. Considering that ketone supplementation inhibits VM-M3 cell proliferation and viability and inhibits HIF-1 signaling in vitro, it is likely that both of these phenomenon contribute to any potential decreased vascularization in the KD+KE treated mouse liver metastases. 130

148 We had hypothesized that we could enhance the anti-cancer efficacy of the KD with ketone supplementation. We found this to be true. While KD-USF, the specific KD used for this study, prolonged mean survival time of VM-M3 mice by approximately 45% (Chapter 5, Figure 5.8), KD+BD and KD+KE treatment prolonged mean survival time by 61.2% and 65.4%, respectively (Figure 4.12). This study strongly suggests that ketone supplemented ketogenic diets can inhibit progression of metastatic cancer in vivo. 4.4 Effect of Chronic Oral Administration of Ketone Supplements in Rats 131

149 Figure Effect of chronic ketone administration on blood glucose in rats. Rats received a daily 5 g/kg dose of water (control), BD, or KE (N 8) via intragastric gavage for 29 days. Blood glucose was significantly reduced in KE-treated, but not BD-treated, rats for approximately 1 hour following ketone supplement administration (*p<0.05, **p<0.01, ***p<0.001; one-way ANOVA). Error bars represent ±SEM. Results were considered significant when p<0.05. Figure Effect of chronic ketone administration on blood βhb in rats. Rats received a daily 5 g/kg dose of water (control), BD, or KE (N 8) via intragastric gavage for 29 days. Blood 132

150 βhb was significantly elevated in both BD-treated and KE-treated rats for approximately 4-8 hours following ketone supplement administration (*p<0.05, **p<0.01, ***p<0.001; one-way ANOVA). Error bars represent ±SEM. Results were considered significant when p<0.05. Figure Correlation between blood glucose and blood βhb following oral ketone supplement administration. Rats received a daily 5 g/kg dose of water (control), BD, or KE (N 8) via intragastric gavage for 29 days. Correlation between blood glucose and blood βhb concentration following acute (week 0; day 0) and chronic (week 4; day 28) ketone supplement administration was determined by linear regression analysis. There was a significant inverse correlation between blood glucose and blood βhb concentration in BD-treated rats at week 4 (*p<0.01), and a strong trend towards an inverse relationship between blood glucose and blood 133

151 in BD-treated rats at week 0 and in KE-treated rats at weeks 0 and 4. Error bars represent ±SEM. Results were considered significant when p<0.05. Figure Effect of chronic ketone administration on blood triglycerides, total cholesterol, HDL, and LDL in rats. Rats received a daily 5 g/kg dose of water (control), BD, or KE (N 8) via intragastric gavage for 29 days. On day 28, blood triglycerides, total cholesterol, and HDL were measured. LDL cholesterol was determined from the measured parameters with the Friedewald equation. There was no significant effect of chronic administration of BD and KE on blood lipid levels (p>0.05; one-way ANOVA). Error bars represent ±SEM. 134

152 Table 4.1. Global metabolomics profiling of serum from rats following chronic administration of KE. Rats received a daily 5 g/kg dose of water (control) or KE (N 8) via intragastric gavage for 29 days. Serum was taken from rats on day 29 and global metabolomics profiling was performed at Metabolon Inc. using gas and liquid-chromatography with tandem mass spectrometry. 155 of 388 known metabolites were significantly changed from control rats (*p<0.05, **p<0.01, ***p<0.001; Welch s two-sample t-test). Metabolites affected by KEtreatment with potential relevance to the anti-cancer efficacy of ketone supplementation are shown. Results were considered significant when p<0.05 and are indicated by solid red or green backgrounds. Metabolites that exhibited a trend of change but were not statistically significant (0.05<p<0.10) are indicated by a shaded red or green background. Super Pathway Sub Pathway Biochemical Name Fold of Change from Control glutathione, oxidized (GSSG) 17.34* Glutathione Metabolism cysteine-glutathione disulfide 2.41** Amino Acid Glutamate Metabolism pyroglutamine 0.42** Polyamine Metabolism spermidine 1.98* DTGTTSEFXEAGGDXR 0.65* Fibrinogen Cleavage Peptide ATTDSDKVDXSXA 0.68 Peptide DSDKVDXSXAR 2.07* Carbohydrate Dipeptide Derivative Pentose Phosphate Pathway carnosine ribose 5-phosphate 3.23* 4.86* anserine ribulose/xylulose 5-phosphate 5.66** 8.18* citrate 1.99*** Energy TCA Cycle alpha-ketoglutarate 1.98** fumarate 1.92* malate 2.03** Ketone Bodies acetoacetate 8.84*** 3-hydroxybutyrate (BHBA) 15.78*** 1-pentadecanoylglycerophosphocholine (15:0) 1.36** 2-palmitoylglycerophosphocholine 0.8* 2-palmitoleoylglycerophosphocholine 0.5** 2-stearoylglycerophosphocholine 0.7** 2-oleoylglycerophosphocholine 0.75** 1-linoleoylglycerophosphocholine (18:2n6) 0.79** 2-linoleoylglycerophosphocholine 0.76* 1-nonadecanoylglycerophosphocholine(19:0) 0.64* Lipid 1-dihomo-linoleoylglycerophosphocholine (20:2n6) 0.67* Lysolipid 1-eicosenoylglycerophosphocholine (20:1n9) 0.6** 1-eicosatrienoylglycerophosphocholine (20:3) 0.68* 1-arachidonoylglycerophosphocholine (20:4n6) 0.88* 2-arachidonoylglycerophosphocholine 0.77* 1-docosapentaenoylglycerophosphocholine (22:5n3) 0.72** 1-palmitoylplasmenylethanolamine oleoylplasmenylethanolamine 1.98* 1-linoleoylglycerophosphoethanolamine 0.66* 1-stearoylglycerophosphoserine 2.89* 1-linoleoylglycerophosphoserine 3.45** alpha-tocopherol 0.55** Tocopherol Metabolism Cofactors and Vitamins beta-tocopherol 0.76** gamma-tocopherol 0.61* alpha-cehc sulfate 0.52** Hemoglobin and Porphyrin Metabolism bilirubin (Z,Z) 0.4** 135

153 Chronic oral administration of ketone supplements lowers blood glucose, elevates blood ketones, and dramatically alters serum metabolites in rats Chronic ketone administration induces ketoadaptation, a physiologic state characterized by a shift away from glucose metabolism and towards fat and ketone body metabolism. There are many metabolic changes with sustained ketosis that may underlie its multifaceted therapeutic potential, so we studied how blood glucose, blood ketones, blood lipids, and other biochemical metabolites are affected by chronic KE administration. Over a 29 day period, rats receiving a daily 5 g/kg dose of KE showed a drop in blood glucose for approximately one hour following administration. In general, this drop in blood glucose became more substantial over the course of the study (Figure 4.13), suggesting that chronic ketosis enhances the hypoglycemic effect of ketone administration. Veech and colleagues have demonstrated that administration of a similar ketone ester simultaneously decreased blood glucose and blood insulin by approximately 50%, suggesting that the hypoglycemic effect of ketones is caused by enhancing insulin sensitivity [11]. Our results support this work, showing that ketone ester supplementation elicits a hypoglycemic effect in vivo. Since glucose is the major energy source for cancer, and its elevation is directly correlated to tumor growth, KE supplementation as a cancer therapy may work in part by inhibiting glucose availability to the tumor. Aside from the known proteinsparing effects of ketones, if ketosis enhances insulin sensitivity, it likely increases glucose uptake by the insulin-sensitive skeletal muscle. Together, these effects would help support muscle tissue health and function, suggesting another possible benefit of KE supplementation in slowing muscle wasting associated with cancer cachexia. Blood βhb levels were elevated for approximately 4 hours following KE administration and 8 hours following BD administration (Figure 4.14), demonstrating the ability of oral ketone 136

154 supplementation to induce sustained ketosis in vivo. It is important to remember, however, that while BD is metabolized to βhb, KE metabolism produces both βhb and AcAc. The commercially available blood ketone meters used in this study offer the most cost feasible method of measuring blood ketone levels, but are only able to measure βhb, not AcAc. However, metabolomics analysis indicated a nearly 9-fold increase in AcAc from KE-treated rats compared to controls (Table 4.1). Thus, total blood ketones was likely higher in KE-treated than BD-treated animals. In general, blood βhb levels decreased more quickly during weeks 1-4 compared to the first administration (week 0) (Figure 4.14). This difference is possibly due to an increase in tissue utilization following ketoadaptation that comes with chronic ketosis; however, it is also possible that excess ketones were excreted in the urine, but this was not measured. Notably, the increase in blood βhb was correlated to the decrease in blood glucose following ketone supplementation (Figure 4.15), further supporting the hypothesis that ketones enhance insulin sensitivity. The rats used in this study had not reached their full adult body size yet, and therefore over the four week study, control animals exhibited changes in their blood lipid profile from baseline associated with normal aging (Figure 4.16). Rats receiving BD and KE demonstrated similar changes in triglycerides, total cholesterol, HDL, and LDL from baseline as control rats, indicating that there was no significant effect of chronic ketone supplementation on blood lipids (Figure 4.16). Although the evidence is controversial and highly dependent on the specific macro and micronutrient composition of the diet, a common concern regarding the KD is its potential negative effect on blood triglyceride and cholesterol levels [31, 32]. Thus, ketone supplementation could provide an alternative method to induce ketosis, or a way to enhance the effects of the KD, without a major influence on the blood lipid profile. 137

155 Global metabolomics profiling of serum from chronic KE-treated rats revealed 155 metabolites which were significantly altered compared to control. A selection of affected metabolites with potential relevance to the anti-cancer effects of ketone supplementation is provided in Table 4.1. These metabolite changes provide preliminary insight into several potential mechanisms of the anti-cancer efficacy of KE supplementation for future studies. Unexpectedly, markers of both oxidative stress (oxidized glutathione, GSSG) and antioxidant capacity (carnosine and anserine) were elevated, suggesting a heightened state of oxidative stress and antioxidant defense with KE supplementation. Generally, ketone metabolism is known to decrease ROS production and enhance endogenous antioxidant capacity [33]. It should be noted, however, that the dose of KE-administration was relatively high, and delivered as a daily bolus. It is likely that delivering a lower dose over a more sustained period of time, such as when mixed into food, would affect this response. As described, ROS production, antioxidant capacity, and redox state dramatically influences cancer progression [34]. Both pro-oxidant and anti-oxidant therapies have shown anti-cancer effects in vitro and in vivo [35]. The altered redox state induced by KE administration likely plays a role in its anti-cancer effects and provides a clear point of interest for future studies into the molecular mechanisms of ketone metabolism in cancer. The antioxidant carnosine was nearly 4-fold higher in KE-treated rats compared to controls (Table 4.1). Carnosine has been investigated as a potential anti-neoplastic therapeutic [36], and has been shown to inhibit cancer cell viability in vitro, and inhibit tumor growth and metastatic spread and increase survival in vivo [36-39]. Carnosine is a known antioxidant, but the extent of its physiological functions remain unknown [40, 41]. One study determined that carnosine inhibits anaerobic glycolysis, which suggests that it may target the Warburg effect in 138

156 cancer [42]. Alteration of redox capacity by carnosine may play a role in its anti-cancer effects as well. KE-induced elevation of carnosine provides an interesting potential mechanism for use of KE supplementation as a cancer therapy. Lysophospholipids (LP) are extracellular lipid mediators that bind to a family of G protein coupled receptors (GPCRs) and affect numerous cellular functions including differentiation, proliferation, survival, adhesion, and invasion [43]. LPs elicit many growth factor-like effects, and not surprisingly, LP signaling is altered in cancer [44, 45]. Studies have found elevated LP variants in the blood and peritoneal fluid of ovarian cancer patients, while LP receptor elevation and signaling helps mediate survival in ovarian cancer cell lines [45]. LP receptor overexpression induces tumorigenesis and metastasis in mice, and knockdown elicits opposite effects [46]. Several lysophospholipids were significantly decreased in KE-treated rats (Table 4.1), offering insight into numerous potential mechanisms by which ketones affect cancer cell growth and survival. In our VM-M3 mouse studies, KE was mixed into the food and fed ad libitum, allowing continual delivery of the KE in smaller doses as the mice fed throughout the day. Compared to a bolus administration, multiple daily doses or inclusion in food would likely provide more sustained beneficial effects. The metabolomics profiling data presented here help us to understand the metabolic consequences of KE administration and offers insight into potential mechanisms of action by which ketone supplementation may inhibit cancer progression for future studies. 139

157 4.5 References for Chapter 4 1. Seyfried TN, Shelton LM: Cancer as a metabolic disease. Nutr Metab (Lond) 2010, 7:7. 2. Skinner R, Trujillo A, Ma X, Beierle E: Ketone bodies inhibit the viability of human neuroblastoma cells. Journal of pediatric surgery 2009, 44(1): Seyfried TN: Cancer as a Metabolic Disease: On the Origin, Management, and Prevention of Cancer. Hoboken, NJ: John Wiley & Sons, In.; Cahill GF, Jr., Veech RL: Ketoacids? Good medicine? Trans Am Clin Climatol Assoc 2003, 114: ; discussion Magee BA, Potezny N, Rofe AM, Conyers RA: The inhibition of malignant cell growth by ketone bodies. Aust J Exp Biol Med Sci 1979, 57(5): Robey IF, Lien AD, Welsh SJ, Baggett BK, Gillies RJ: Hypoxia-inducible factor- 1alpha and the glycolytic phenotype in tumors. Neoplasia 2005, 7(4): Chen L, Shi Y, Yuan J, Han Y, Qin R, Wu Q, Jia B, Wei B, Wei L, Dai G et al: HIF-1 Alpha Overexpression Correlates with Poor Overall Survival and Disease-Free Survival in Gastric Cancer Patients Post-Gastrectomy. PLoS One 2014, 9(3):e Miyake K, Yoshizumi T, Imura S, Sugimoto K, Batmunkh E, Kanemura H, Morine Y, Shimada M: Expression of hypoxia-inducible factor-1alpha, histone deacetylase 1, and metastasis-associated protein 1 in pancreatic carcinoma: correlation with poor prognosis with possible regulation. Pancreas 2008, 36(3):e Semenza GL: Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics. Oncogene 2010, 29(5): Pal I, Mandal M: PI3K and Akt as molecular targets for cancer therapy: current clinical outcomes. Acta pharmacologica Sinica 2012, 33(12): Kashiwaya Y, Pawlosky R, Markis W, King MT, Bergman C, Srivastava S, Murray A, Clarke K, Veech RL: A ketone ester diet increases brain malonyl-coa and Uncoupling proteins 4 and 5 while decreasing food intake in the normal Wistar Rat. The Journal of biological chemistry 2010, 285(34): Paoli A, Rubini A, Volek JS, Grimaldi KA: Beyond weight loss: a review of the therapeutic uses of very-low-carbohydrate (ketogenic) diets. Eur J Clin Nutr 2013, 67(8): Rebucci M, Michiels C: Molecular aspects of cancer cell resistance to chemotherapy. Biochemical pharmacology 2013, 85(9): Berrigan D, Perkins S, Haines D, Hursting S: Adult-onset calorie restriction and fasting delay spontaneous tumorigenesis in p53-deficient mice. Carcinogenesis 2002, 23(5): Hursting S, Smith S, Lashinger L, Harvey A, Perkins S: Calories and carcinogenesis: lessons learned from 30 years of calorie restriction research. Carcinogenesis 2010, 31(1): Mukherjee P, Abate LE, Seyfried TN: Antiangiogenic and proapoptotic effects of dietary restriction on experimental mouse and human brain tumors. Clin Cancer Res 2004, 10(16): Shelton LM, Huysentruyt LC, Mukherjee P, Seyfried TN: Calorie restriction as an antiinvasive therapy for malignant brain cancer in the VM mouse. ASN Neuro 2010, 2(3):e

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160 CHAPTER 5: COMBINATION METABOLIC THERAPY ELICITS POTENT ANTI- CANCER EFFECTS IN VM-M3 CANCER IN VITRO AND IN VIVO 5.1 Chapter Synopsis Here we describe the effects of combination ketogenic diet, ketone supplementation, and hyperbaric oxygen therapy on VM-M3 cancer in vitro and in vivo. In the previous chapters, we demonstrated that these therapies were effective to varying degrees as stand-alone treatments; however, combining them elicited potent, synergistic anti-cancer effects in the VM-M3 model that provided the most significant response of the multiple therapeutic regimens tested in this study. Proliferation and viability was decreased and ROS production increased in VM-M3 cells receiving combination low glucose, βhb, and HBOT. VM-M3 mice receiving combination KD+KE+HBOT therapy had a significant reduction in tumor growth rate and metastatic spread, metastatic tumor vascularization, and lived twice as long as control animals. We hypothesize that this novel combination therapy could provide an effective stand-alone or adjuvant treatment option for aggressive cancers of multiple cancer types. Portions of this chapter have been taken from a manuscript accepted with revisions in PLOS One: Poff A, Ward N, Seyfried T, Arnold P, D Agostino D. (2014) Non-toxic metabolic management of metastatic cancer in VM mice: Novel combination of ketogenic diet, ketone supplementation, and hyperbaric oxygen therapy. A copy of this article may be found in 143

161 Appendix C. See Appendix B for copyright permissions. Materials and methods for the studies presented in this chapter can be found in Appendix A. 5.2 Combination Metabolic Therapy Inhibits VM-M3 Cancer in Vitro and in Vivo As described in chapter 2, the KD, ketone supplementation, and HBOT are three metabolic therapies which target overlapping metabolic pathways that are critical to cancer progression and are especially prominent in metastatic cells. In chapter 3, we demonstrated that ketosis and HBOT created a physiological environment that was not supportive of metastatic cancer growth and survival in the VM-M3 model of metastatic cancer. In chapter 4, we demonstrated that supplemental ketone administration could induce a state of therapeutic ketosis, inhibit tumor growth and prolong survival as a stand-alone therapy, and improve the anti-cancer response of the KD in this model. In hopes of further understanding the potential use of these metabolic therapies, and in an attempt to pursue the maximal therapeutic response of the novel combination of ketosis and hyperbaric oxygen, we investigated the effects of combining the KD, KE, and HBOT in VM-M3 mice in vivo and mimicking this physiological environment VM-M3 cells in vitro. 144

162 Figure 5.1. Combination metabolic therapy decreases VM-M3 cell proliferation in vitro. VM-M3 cells were grown in conditions mimicking metabolic therapy, including combinations of high to low glucose media, ketone supplementation, and daily HBOT sessions. Cell density was measured with trypan blue hemocytometry to produce a growth curve over 96 hours. (A) Proliferation was inhibited when treated with decreasing glucose concentration and adjuvant HBOT. (B) Combining low glucose, ketone supplementation, and HBOT resulted in a marked decrease in cell proliferation which was significant from control at all time points tested. (p<0.05; one-way ANOVA). Cells receiving combination therapy had a significant decrease in proliferation compared to cells treated with LG only at 24, 48, and 72 hours (p<0.05; one-way ANOVA), and exhibited a non-significant trend of decreased proliferation compared to LG + HBOT treated cells for the duration of the growth curve. Results were considered significant when p<0.05. Error bars represent ±SEM. 145

163 Figure 5.2. Combination metabolic therapy decreases VM-M3 cell viability in vitro. VM- M3 cells were treated with high (control; 25mM) or low (LG; 3mM) glucose 24 hours, with or without one session of hyperbaric oxygen. Viability was measured with calcein AM and Ethd-1 fluorescence microscopy. (A-B) LG and LG+βHB+HBOT treatment decreased viability compared to control (***p<0.001; one-way ANOVA). Results were considered significant when p<0.05. Error bars represent ±SEM. 146

164 Figure 5.3. Metabolic therapy increases ROS production and decreases HIF-1α expression in VM-M3 cells. (A-B) VM-M3 cells were treated for 24 hours with individual or combination metabolic therapies: LG, βhb, HBOT, or LG+βHB+HBOT. Superoxide production was measured with DHE fluorescence microscopy and was significantly increased with HBOT (***p<0.001; one-way ANOVA). (C-D) VM-M3 cells were treated for 7 days in control media with either ketone supplementation (βhb) or daily hyperbaric oxygen sessions (HBOT). HIF-1α expression was measured by western blot analysis and was significantly decreased with βhb and HBOT treatment (***p<0.001; one-way ANOVA). Results were considered significant when p<0.05. Error bars represent ±SEM. 147

165 Figure 5.4. Blood glucose, βhb, and body weight of VM-M3 survival study mice prior to significant disease onset. (A-C) KD+KE treated mice had significantly decreased blood glucose and body weight and increased blood βhb compared to control mice by day 7 (**p<0.01, ***p<0.001; one-way ANOVA). KD+KE and KD+KE+HBOT mice exhibited increased blood βhb compared to controls by day 7 (***p<0.001; one-way ANOVA). KD+KE mice had a significant reduction in body weight by day 7 (***p<0.001; one-way ANOVA). Results were considered significant when p<0.05. Error bars represent ±SEM. 148

166 Figure 5.5. Combination metabolic therapy slows tumor growth and inhibits metastatic spread. (A) In vivo bioluminescence imaging of representative animals from each treatment group 3 weeks post tumor cell inoculation. Treated mice exhibited reduced tumor burden and metastatic spread. (B) Whole animal bioluminescence (photons/sec) was tracked over time as a measure of tumor growth rate. In order to avoid misleading representation of tumor burden, data for treatment groups where 50% of animals had succumbed to disease progression are no longer shown. Tumor bioluminescence was highly variable, but treated animals demonstrated a notable trend of slower tumor growth compared to controls throughout the study. At week 3, KD+KE+HBOT mice had significantly less bioluminescence than control animals (**p<0.01; one-way ANOVA). (C-E) Bioluminescence of key metastatic regions 21 days after tumor cell inoculation demonstrated a marked reduction in metastatic tumor burden and spread for treated animals. KD+KE+HBOT mice had significantly less metastatic spread to the brain, lung, and liver compared to controls at 3 weeks (*p<0.05, **p<0.01; one-way ANOVA). Error bars represent ±SEM. Results were considered significant when p<

167 Figure 5.6. Effect of KD+KE+HBOT treatment on metastatic spread by ex vivo bioluminescence. VM-M3 mice received either standard rodent chow (control) or KD+KE+HBOT treatment for 21 days following tumor cell inoculation. Organs were harvested and bioluminescent signal recorded as a measure of metastatic spread. KD+KE+HBOT treated mice exhibited significantly decreased bioluminescence in the brain, kidneys, spleen, lungs, 150

168 adipose, and liver (***p<0.001; unpaired t-test). Error bars represent ±SEM. Results were considered significant when p<0.05. Figure 5.7. Effect of KD+KE+HBOT treatment on metastatic liver tumor size and vascularization. Morphology and vascularization of KD+KE+HBOT-treated mouse liver tumors were analyzed by H&E staining and CD31 immunohistochemistry. Tumors from KD+KE+HBOT-treated mice were smaller with more well-defined borders (A, C) and exhibited a significant reduction in vascularization (B, D, E) from controls (*p<0.05; unpaired t-test). Error bars represent ±SEM. 151

169 Figure 5.8. Combination metabolic therapy extends survival time in mice with metastatic cancer. (A) Kaplan-Meier survival curve of study groups. KD, KD+KE, and KD+KE+HBOT treated mice demonstrated prolonged survival compared to controls (p=0.03, p=0.009, and p<0.0001, respectively; log-rank Mantel-Cox test for survival distribution). (B) Cohort size (N), mean survival (days), and percent increase in mean survival time from control. KD, KD+KE, and KD+KE+HBOT treated mice exhibited a 44.6%, 65.4%, and 103.2% increase in mean survival time compared to controls, respectively (*p<0.05, **p<0.01, ***p<0.001; one-way ANOVA). Results were considered significant when p<0.05. Combination metabolic therapy inhibits VM-M3 cancer in vitro and in vivo Metastasis remains the largest obstacle in identifying effective therapies for the long-term management of or a cure for cancer. A lack of animal models which reflect the metastatic phenotype prevents major improvements in patient care. The major goal of this study was to investigate the anti-cancer efficacy of a novel combination of metabolic therapies the ketogenic diet, ketone supplementation, and hyperbaric oxygen in a mouse model of aggressive metastatic cancer that recapitulates much of the metastatic phenotype seen in invasive human 152

170 cancers [1-4]. Individually, these therapies inhibited proliferation and viability in vitro, slowed disease progression in vivo, and when combined, elicited potent anti-cancer effects by inhibiting metastatic spread and doubling the survival time of mice with systemic, metastatic disease. We have previously shown that ketone supplementation and combination ketogenic diet with HBOT slowed cancer progression in the VM-M3 model of metastatic cancer [5, 6]. We hypothesized that combining these therapeutic regimens could provide an even more significant response in this model. The KD has been used clinically to treat pediatric refractory epilepsy for nearly a century, and is known to be a safe and feasible option for patients [7, 8]. HBOT is an approved therapy for several disease states, including decompression sickness, carbon monoxide poisoning, and radionecrosis [9]. Its use and safety is well-characterized and understood [9, 10]. Exogenous ketone ester supplementation is a novel therapy that is also safe and feasible [11, 12]. When ketone supplementation is administered properly, blood ketone levels remain in normal physiological levels and do not approach the dangerous levels associated with diabetic ketoacidosis (up to 20 mm). Ketone supplementation can be used, however, to elevate blood ketone levels to a therapeutic range of 2-7mM [6], which is reported to be beneficial in a vast array of disease states, including epilepsy, Alzheimer s disease, and other neurological disorders [7, 13-19]. Importantly, these treatments represent non-toxic alternative or adjuvant therapies, which could be readily implemented clinically if their effects hold up in human trials. The VM-M3 model exhibits rapid, systemic metastasis following s.c. inoculation [1]. As demonstrated by the in vivo bioluminescent imaging (Figure 1), primary tumor growth and pattern of metastatic spread can be highly variable, such as is seen in many human metastatic cancers. The KD, ketone supplementation, and HBOT induced a trend of slowed tumor growth rate in vivo, which was significant in mice receiving combination therapy (Figure 5.5) [5, 6]. 153

171 The effect of these metabolic therapies on proliferation and viability in vitro mimicked the effects seen in vivo. Both βhb and HBOT significantly decreased VM-M3 cell proliferation, and this effect was enhanced by decreasing blood glucose (Figure 5.1) [6]. We showed previously that βhb decreased VM-M3 cell viability, even in the presence of high glucose, but this effect was enhanced by lowering glucose concentration [6]. HBOT alone did not increase survival time in vivo, and although there was a trend of decreased viability in vitro, this was not statistically significant; however, combining HBOT with ketosis elicited potent anti-cancer effects in mice and cells (Figures 3.1, 5.8) [5]. Taken together, these studies suggest that combining therapeutic ketosis with HBOT creates a unique physiologic and metabolic environment that is not conducive to rapid cancer cell proliferation and survival. Blood glucose and body weight decreased and blood βhb increased in mice receiving KD+KE therapy by day 7, prior to the onset of significant disease progression (Figure 5.4). As would be expected, blood glucose was lower and blood βhb was more elevated in KD+KE mice compared to KD alone. KD+KE mice did lose approximately 20% of their initial body weight, suggesting a potential therapeutic contribution from calorie restriction. However, we previously showed that the anti-cancer effects of KE supplementation was not due to CR in body weight matched animals (Figure 4.4) [6]. Interestingly, at day 7, KD+KE+HBOT treated mice had increased blood βhb, but blood glucose and body weight were not significantly different from controls. The discrepancy between these two groups receiving the same dietary therapy is unclear. Alteration of tissue oxygenation is known to affect metabolic capacity and blood metabolite levels [20]. Blood glucose decreases immediately following HBOT sessions in both animals and humans [21]. This may be due to an increase in insulin secretion and tissue glucose utilization which has been reported with HBOT [22-25]. In our study, blood metabolites were 154

172 measured several hours after HBOT. Perhaps HBOT-induced glucose utilization by the brain and peripheral tissues stimulated gluconeogenesis or feeding behavior in the VM-M3 mice. This could possibly explain why glucose was not decreased at the time of blood measurement and why these animals did not lose weight as the KD+KE mice did. Similarly, HBOT has been shown to enhance weight gain in head and neck cancer patients following surgery and radiation [26]. In order to provide a more in depth understanding of the effect of combination KD+KE+HBOT therapy on metastatic spread, ex vivo bioluminescent imaging was performed on organs harvested from control and treated animals 21 days post tumor cell implantation (Figure 5.6). The results echoed what was observed with in vivo bioluminescent imaging of metastatic regions (Figure 5.5). Control animals exhibited significant metastatic spread to the brain, lungs, liver, and adipose tissue, and a small degree of metastatic spread to the kidneys and spleen (Figure 5.6). Metastatic spread to all harvested organs was significantly reduced, or below detectable threshold, in KD+KE+HBOT treated animals (Figure 5.6). Morphological and immunohistochemical analysis of livers from KD+KE+HBOT treated mice revealed only micrometastatic lesions with little notable vascularization compared to controls (Figure 5.7). These data suggest that combination therapy inhibits metastatic spread and metastatic tumor vascularization, which likely contributes to its potent ability to prolong survival time in VM-M3 mice (Figure 5.4). As expected, HBOT increased ROS production in VM-M3 cells when administered alone, or in combination with LG+βHB (Figure 5.3). Although cancer cells thrive in a state of elevated basal ROS production, they are very sensitive to modest increases or decreases in ROS or alterations in redox or antioxidant state [27, 28]. Furthermore, the differential susceptibility of 155

173 cancer and normal cells to glucose deprivation has been shown to be mediated by superoxide and hydrogen peroxide production and signaling [29, 30]. This may help explain why combining HBOT with glucose restriction, such as with the KD, elicit synergistic effects in vivo [5]. Combining ketosis with hyperbaric oxygen may specifically target cancer metabolism while supporting the health of normal tissues and preventing the many negative symptoms of disease and standard care, like metabolic syndrome and radiation necrosis [31, 32]. We and others have shown that many cancers are unable to effectively metabolize ketones; however, they are an efficient and protective energy source for healthy tissues [6, 19, 33]. Ketone metabolism decreases ROS production and increases the endogenous antioxidant profile of cells which consume them [19, 34]. Since many therapies, including chemotherapy, radiation, and HBOT, work in part by increasing ROS production in the tumor, simultaneous ketone metabolism by surrounding healthy tissue may work to prevent toxic side effects in these tissues. Both ketosis and HBOT have been shown to sensitize tumors to radiation and chemotherapy [35-38]. Concurrent administration of these therapies may allow for the delivery of standard care at reduced doses, lessening toxic side effects and supporting the health of the individual. Importantly, βhb did not decrease ROS production in the VM-M3 cells (Figure 5.3), further supporting the notion that these cancer cells are not able to metabolize ketones as healthy cells do. Both βhb and HBOT significantly decreased HIF-1α expression in VM-M3 cells (Figure 5.3), suggesting one possible mechanism by which these therapies exert their anti-cancer effects. The HIF-1 transcription factor is overly activated in most cancers, and HIF-1α expression correlates strongly with cancer progression and poor prognosis in humans [39-41]. HIF-1 signaling increases the expression of numerous genes which promote survival, proliferation, 156

174 angiogenesis, and mediate the Warburg effect in tumors [41, 42]. Many chemotherapeutic agents work in part by inhibiting HIF-1α expression, including the BCR-ABL inhibitor imatinib (Gleevac), the EGF receptor inhibitor gefitinib (Iressa), and the HER2 neu inhibitor trastuzumab (Herceptin), among others [43]. Ketosis and HBOT will likely elicit similar effects on HIF-1 signaling to these conventional therapeutics, but with significantly less adverse side effects, and may provide potent synergistic effects when used in conjunction with standard care. There is understandable concern regarding the manipulation of diet in vulnerable cancer patients who may find it difficult to consume adequate calories [44]. If these adjuvant therapies allow for decreased yet effective doses of chemotherapy and radiation, patients will likely have an easier time consuming enough calories to support their metabolic needs. Indeed, late-stage patients given the KD report improved quality of life, and small clinical studies suggest that many patients are willing and able to implement the dietary restrictions of the ketogenic diet [37, 45-47]. Simultaneous administration of a ketone supplement such as the ketone ester could potentially provide the same benefit of a KD without the need for such restrictive food intake [6]. Furthermore, ketone metabolism is a protein sparing process [48]. The human brain evolved to metabolize ketones as a way to spare muscle protein during prolonged periods of food deprivation [18]. Ketogenic diets promote weight loss in overweight patients, but they are also known to prevent muscle wasting during physical stress, energy restriction, or starvation [49-52]. In a small study on humans with advanced cancer and cachexia, patients maintained a positive nitrogen balance and gained weight when given a ketone-supplemented ketogenic diet [53]. Indeed, an elevation in blood ketones decreases amino acid release from muscle and decreases hepatic gluconeogenesis of amino acids [19]. In a mouse model of cancer cachexia, mice given a KD had significantly smaller tumors but did not lose as much body weight as control animals 157

175 [54]. Furthermore, control animals had high levels of circulating catabolic factors which elicit triglyceride and amino acid release, but this was inhibited by βhb [53, 54]. Similar studies showed that in mice with a colon cancer that elicits a severe cachexic phenotype, the brain greatly increased expression of 3-oxoacid CoA-transferase, a ketone utilizing enzyme, and relied more heavily on βhb for energy [55]. Enhancing ketone availability as an alternative fuel to the brain and other healthy tissues with the ketogenic diet and/or ketone supplementation will likely support their function during the cachexic condition. There is potential for synergy between these adjuvant metabolic therapies and standard care [47, 56-58]. Scheck and colleagues reported a significant synergistic effect of combining the KD with radiation therapy in a mouse model of brain cancer [35]. Other studies have demonstrated increased efficacy by pairing the KD with chemotherapy or pharmacological agents which target metabolism [37, 38]. HBOT has been investigated as a radiosensitizer in a number of clinical studies [57, 59]. Meta-analyses conclude that HBOT appears to have a neutral or inhibiting effect on cancer progression, depending on cancer type [36, 57, 60]. When administered concurrently with radiation, HBOT potentiates efficacy, but may also increase soft tissue necrosis in nearby healthy tissue [36]. Studies indicate that this adverse effect can be mitigated by delivering HBOT immediately prior to, rather than concurrent with, radiation therapy [61]. Indeed, in this study, human glioma patients receiving HBOT immediately prior to radiation therapy had a median survival time of 24 months, compared to 12 months in patients receiving radiation alone, and did not experience significant side effects [61]. As previously discussed, we hypothesize that ketone metabolism by the healthy tissue may also mitigate adverse effects and perhaps potentiate the efficacy of HBOT and/or standard care. We previously showed that HBOT alone did not affect survival in the VM-M3 model, but 158

176 dramatically slowed progression when administered with a KD [5]. Perhaps other studies that found no effect of HBOT could have revealed therapeutic potential by coupling this therapy with ketosis, either by the KD or ketone supplementation. Despite 50 years of intensive research, cancer remains the second leading cause of death in the United States. This is largely due to a lack of therapies effective for long-term management of metastatic disease, a dilemma underlined by inadequate preclinical models. In this study, we evaluated the efficacy of a novel, non-toxic combination metabolic therapy the ketogenic diet, ketone supplementation, and hyperbaric oxygen in a mouse model of metastatic disease which we believe reliably reflects the human metastatic phenotype [1-4]. Our study suggests that this combination therapy presents a safe, cost-effective, adjuvant to standard care which could provide novel therapeutic options for patients with late-stage cancer. We believe that it is critically important to evaluate non-toxic adjuvant therapies like these, and to look for synergistic potential with other metabolic therapies and standard care. It is possible that we already possess therapeutic options which could significantly improve patient prognosis or outcome when delivered in the appropriate combinations. 5.3 References for Chapter 5 1. Huysentruyt LC, Mukherjee P, Banerjee D, Shelton LM, Seyfried TN: Metastatic cancer cells with macrophage properties: evidence from a new murine tumor model. International journal of cancer 2008, 123(1): Huysentruyt LC, Shelton LM, Seyfried TN: Influence of methotrexate and cisplatin on tumor progression and survival in the VM mouse model of systemic metastatic cancer. Int J Cancer 2010, 126(1): Shelton LM, Huysentruyt LC, Seyfried TN: Glutamine targeting inhibits systemic metastasis in the VM-M3 murine tumor model. Int J Cancer 2010, 127(10):

177 4. Shelton LM, Mukherjee P, Huysentruyt LC, Urits I, Rosenberg JA, Seyfried TN: A novel pre-clinical in vivo mouse model for malignant brain tumor growth and invasion. J Neurooncol 2010, 99(2): Poff AM, Ari C, Seyfried TN, D'Agostino DP: The ketogenic diet and hyperbaric oxygen therapy prolong survival in mice with systemic metastatic cancer. PLoS One 2013, 8(6):e Poff AM, Ari C, Arnold P, Seyfried TN, D'Agostino DP: Ketone supplementation decreases tumor cell viability and prolongs survival of mice with metastatic cancer. Int J Cancer Paoli A, Rubini A, Volek JS, Grimaldi KA: Beyond weight loss: a review of the therapeutic uses of very-low-carbohydrate (ketogenic) diets. Eur J Clin Nutr 2013, 67(8): Groesbeck DK, Bluml RM, Kossoff EH: Long-term use of the ketogenic diet in the treatment of epilepsy. Dev Med Child Neurol 2006, 48(12): Thom S: Hyperbaric oxygen: its mechanisms and efficacy. Plastic and reconstructive surgery 2011, 127 Suppl Gill AL, Bell CNA: Hyperbaric oxygen: its uses, mechanisms of action and outcomes. QJM 2004, Clarke K, Tchabanenko K, Pawlosky R, Carter E, Knight NS, Murray AJ, Cochlin LE, King MT, Wong AW, Roberts A et al: Oral 28-day and developmental toxicity studies of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate. Regul Toxicol Pharmacol 2012, 63(2): Clarke K, Tchabanenko K, Pawlosky R, Carter E, Todd King M, Musa-Veloso K, Ho M, Roberts A, Robertson J, Vanitallie TB et al: Kinetics, safety and tolerability of (R)-3- hydroxybutyl (R)-3-hydroxybutyrate in healthy adult subjects. Regul Toxicol Pharmacol 2012, 63(3): Kashiwaya Y, Bergman C, Lee JH, Wan R, King MT, Mughal MR, Okun E, Clarke K, Mattson MP, Veech RL: A ketone ester diet exhibits anxiolytic and cognition-sparing properties, and lessens amyloid and tau pathologies in a mouse model of Alzheimer's disease. Neurobiol Aging 2013, 34(6): D'Agostino D, Pilla R, Held H, Landon CS, Ari C, Arnold P, Dean JB: Development and testing of ketone esters for prevention of CNS oxygen toxicity seizures. In: Third International Symposium on Dietary Therapies for Epilepsy & Other Neurological Disorders. Chicago, IL; D'Agostino DP, Pilla R, Held HE, Landon CS, Puchowicz M, Brunengraber H, Ari C, Arnold P, Dean JB: Therapeutic ketosis with ketone ester delays central nervous system oxygen toxicity seizures in rats. Am J Physiol Regul Integr Comp Physiol Krikorian R, Shidler MD, Dangelo K, Couch SC, Benoit SC, Clegg DJ: Dietary ketosis enhances memory in mild cognitive impairment. Neurobiol Aging 2012, 33(2):425 e Gasior M, Rogawski MA, Hartman AL: Neuroprotective and disease-modifying effects of the ketogenic diet. Behav Pharmacol 2006, 17(5-6): Cahill GF, Jr., Veech RL: Ketoacids? Good medicine? Trans Am Clin Climatol Assoc 2003, 114: ; discussion

178 19. Veech RL: The therapeutic implications of ketone bodies: the effects of ketone bodies in pathological conditions: ketosis, ketogenic diet, redox states, insulin resistance, and mitochondrial metabolism. Prostaglandins, leukotrienes, and essential fatty acids 2004, 70(3): Boveris A, Costa LE, Poderoso JJ, Carreras MC, Cadenas E: Regulation of mitochondrial respiration by oxygen and nitric oxide. Ann N Y Acad Sci 2000, 899: Peleg RK, Fishlev G, Bechor Y, Bergan J, Friedman M, Koren S, Tirosh A, Efrati S: Effects of hyperbaric oxygen on blood glucose levels in patients with diabetes mellitus, stroke or traumatic brain injury and healthy volunteers: a prospective, crossover, controlled trial. Diving Hyperb Med 2013, 43(4): Dedov, II, Lukich VL, Bol'shakova TD, Gitel EP, Dreval AV: [Effect of hyperbaric oxygenation on residual insulin secretion in patients with diabetes mellitus type 1]. Probl Endokrinol (Mosk) 1987, 33(4): Contreras FL, Kadekaro M, Eisenberg HM: The effect of hyperbaric oxygen on glucose utilization in a freeze-traumatized rat brain. J Neurosurg 1988, 68(1): Torbati D: Central nervous system glucose utilization rate during oxygen-induced respiratory changes at 2 atmospheres oxygen in the rat. J Neurol Sci 1986, 76(2-3): Bassett DJ, Fisher AB: Glucose metabolism in rat lung during exposure to hyperbaric O2. J Appl Physiol Respir Environ Exerc Physiol 1979, 46(5): Schoen PJ, Raghoebar GM, Bouma J, Reintsema H, Vissink A, Sterk W, Roodenburg JL: Rehabilitation of oral function in head and neck cancer patients after radiotherapy with implant-retained dentures: effects of hyperbaric oxygen therapy. Oral oncology 2007, 43(4): Schumacker P: Reactive oxygen species in cancer cells: live by the sword, die by the sword. Cancer cell 2006, 10: Wang J, Yi J: Cancer cell killing via ROS: to increase or decrease, that is the question. Cancer Biol Ther 2008, 7(12): Aykin-Burns N, Ahmad I, Zhu Y, Oberley L, Spitz D: Increased levels of superoxide and H2O2 mediate the differential susceptibility of cancer cells versus normal cells to glucose deprivation. The Biochemical journal 2009, 418: Spitz DR, Sim JE, Ridnour LA, Galoforo SS, Lee YJ: Glucose deprivation-induced oxidative stress in human tumor cells. A fundamental defect in metabolism? Ann N Y Acad Sci 2000, 899: Tonorezos ES, Jones LW: Energy balance and metabolism after cancer treatment. Semin Oncol 2013, 40(6): Feldmeier JJ: Hyperbaric oxygen therapy and delayed radiation injuries (soft tissue and bony necrosis): 2012 update. Undersea Hyperb Med 2012, 39(6): Barbara AM, Nicholas P, Allan MR, Robert AJC: THE INHIBITION OF MALIGNANT CELL GROWTH BY KETONE BODIES. Australian Journal of Experimental Biology and Medical Science 1979, Veech RL, Chance B, Kashiwaya Y, Lardy HA, Cahill GF, Jr.: Ketone bodies, potential therapeutic uses. IUBMB Life 2001, 51(4):

179 35. Abdelwahab M, Fenton K, Preul M, Rho J, Lynch A, Stafford P, Scheck A: The ketogenic diet is an effective adjuvant to radiation therapy for the treatment of malignant glioma. PLoS One 2012, Moen I, Stuhr LE: Hyperbaric oxygen therapy and cancer-a review. Targeted oncology 2012, 7: Zuccoli G, Marcello N, Pisanello A, Servadei F, Vaccaro S, Mukherjee P, Seyfried TN: Metabolic management of glioblastoma multiforme using standard therapy together with a restricted ketogenic diet: Case Report. Nutr Metab (Lond) 2010, 7: Marsh J, Mukherjee P, Seyfried T: Drug/diet synergy for managing malignant astrocytoma in mice: 2-deoxy-D-glucose and the restricted ketogenic diet. Nutr Metab (Lond) 2008, 5: Chen L, Shi Y, Yuan J, Han Y, Qin R, Wu Q, Jia B, Wei B, Wei L, Dai G et al: HIF-1 Alpha Overexpression Correlates with Poor Overall Survival and Disease-Free Survival in Gastric Cancer Patients Post-Gastrectomy. PLoS One 2014, 9(3):e Miyake K, Yoshizumi T, Imura S, Sugimoto K, Batmunkh E, Kanemura H, Morine Y, Shimada M: Expression of hypoxia-inducible factor-1alpha, histone deacetylase 1, and metastasis-associated protein 1 in pancreatic carcinoma: correlation with poor prognosis with possible regulation. Pancreas 2008, 36(3):e Robey IF, Lien AD, Welsh SJ, Baggett BK, Gillies RJ: Hypoxia-inducible factor- 1alpha and the glycolytic phenotype in tumors. Neoplasia 2005, 7(4): Semenza GL: HIF-1 mediates metabolic responses to intratumoral hypoxia and oncogenic mutations. J Clin Invest 2013, 123(9): Semenza GL: Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics. Oncogene 2010, 29(5): Hutton JL, Martin L, Field CJ, Wismer WV, Bruera ED, Watanabe SM, Baracos VE: Dietary patterns in patients with advanced cancer: implications for anorexiacachexia therapy. The American journal of clinical nutrition 2006, 84(5): Schmidt M, Pfetzer N, Schwab M, Strauss I, Kämmerer U: Effects of a ketogenic diet on the quality of life in 16 patients with advanced cancer: A pilot trial. Nutr Metab (Lond) 2011, 8(1): Fine E, Segal-Isaacson C, Feinman R, Herszkopf S, Romano M, Tomuta N, Bontempo A, Negassa A, Sparano J: Targeting insulin inhibition as a metabolic therapy in advanced cancer: a pilot safety and feasibility dietary trial in 10 patients. Nutrition 2012, 28(10): Champ CE, Palmer JD, Volek JS, Werner-Wasik M, Andrews DW, Evans JJ, Glass J, Kim L, Shi W: Targeting metabolism with a ketogenic diet during the treatment of glioblastoma multiforme. Journal of neuro-oncology 2014, 117(1): Wu GY, Thompson JR: The effect of ketone bodies on alanine and glutamine metabolism in isolated skeletal muscle from the fasted chick. The Biochemical journal 1988, 255(1): Manninen AH: Very-low-carbohydrate diets and preservation of muscle mass. Nutr Metab (Lond) 2006, 3: Cahill G: Fuel metabolism in starvation. Annual review of nutrition 2006, 26: Volek J, Sharman M, Love D, Avery N, Gómez A, Scheett T, Kraemer W: Body composition and hormonal responses to a carbohydrate-restricted diet. Metabolism: clinical and experimental 2002, 51(7):

180 52. Paoli A, Grimaldi K, D'Agostino D, Cenci L, Moro T, Bianco A, Palma A: Ketogenic diet does not affect strength performance in elite artistic gymnasts. Journal of the International Society of Sports Nutrition 2012, 9(1): Fearon KC, Borland W, Preston T, Tisdale MJ, Shenkin A, Calman KC: Cancer cachexia: influence of systemic ketosis on substrate levels and nitrogen metabolism. The American journal of clinical nutrition 1988, 47(1): Tisdale MJ, Brennan RA, Fearon KC: Reduction of weight loss and tumour size in a cachexia model by a high fat diet. Br J Cancer 1987, 56(1): Mulligan HD, Tisdale MJ: Metabolic substrate utilization by tumour and host tissues in cancer cachexia. The Biochemical journal 1991, 277 ( Pt 2): Meijer TW, Kaanders JH, Span PN, Bussink J: Targeting hypoxia, HIF-1, and tumor glucose metabolism to improve radiotherapy efficacy. Clin Cancer Res 2012, 18(20): Al-Waili NS, Butler GJ, Beale J, Hamilton RW, Lee BY, Lucas P: Hyperbaric oxygen and malignancies: a potential role in radiotherapy, chemotherapy, tumor surgery and phototherapy. Med Sci Monit 2005, 11(9):RA Bennewith K, Dedhar S: Targeting hypoxic tumour cells to overcome metastasis. BMC Cancer 2011, 11: Bennett M, Feldmeier J, Smee R, Milross C: Hyperbaric oxygenation for tumour sensitisation to radiotherapy: a systematic review of randomised controlled trials. Cancer treatment reviews 2008, 34(7): Daruwalla J, Christophi C: Hyperbaric oxygen therapy for malignancy: a review. World journal of surgery 2006, 30: Kohshi K, Kinoshita Y, Imada H, Kunugita N, Abe H, Terashima H, Tokui N, Uemura S: Effects of radiotherapy after hyperbaric oxygenation on malignant gliomas. Br J Cancer 1999, 80(1-2):

181 CHAPTER 6: DISCUSSION IMPLICATIONS FOR CANCER THERAPY 6.1 Chapter Synopsis Here we describe the major findings of this work and discuss its overarching implications for cancer therapy. A detailed discussion of the data has been provided in the previous chapters. 6.2 Implications for Cancer Therapy The major goal of this dissertation work was to determine the anti-cancer efficacy of ketosis and HBO therapies in the VM-M3 model of metastatic cancer in vivo and in vitro, and to gather preliminary information on potential mechanisms of action for future studies. These therapies were effective individually to varying extents, but combining a ketone-supplemented ketogenic diet with concurrent HBOT elicited the most significant response in this model. Our work strongly suggests that inducing ketosis in conjunction with HBOT creates a physiological environment that is unfavorable to the progression and survival of even a highly aggressive and malignant cancer like VM-M3. Importantly, this therapy targets aspects of cancer metabolism which are nearly universal in human cancers. Hypoxia is a consistent feature of solid tumors; it is present even in relatively small tumors, and its prevalence only increases with tumor growth [1]. The Warburg effect is also observed in most, if not all, cancers, and its expression also enhances with tumor progression and invasive potential [2]. Therefore, we expect that this 164

182 metabolic therapy would be effective to varying degrees against most cancer types, especially those that form solid tumors and/or are FDG-PET positive. As described in chapter 2, this specific combination of therapies has the potential to target several malignant processes simultaneously, including inhibiting HIF-1 and insulin signaling as well as inhibiting inflammation, angiogenesis, lactate export, and histone deacetylase activity, altering ROS production and redox status, and modulating markers of EMT and the metastatic process. This is very different from the use of targeted drug therapies which act specifically on a single genetic mutation or oncogene pathway. Because of the variability of gene mutations even within a single tumor, and the remarkable mutability of the cancer genome, it is unlikely that even a cocktail of targeted therapies alone would successfully eliminate all cells of an aggressive or metastatic tumor [3]. This appears to be true, as clinical resistance develops against most chemotherapies, and metastatic tumors often recur following a period of remission. In contrast, dysregulated metabolism appears to be a universal feature of cancer cells and for this reason has been called cancer s Achilles heel [4]. Perhaps most importantly, the Warburg effect is especially critical for the function and pluripotency of cancer stem cells, the specific subset of tumor cells which escape treatment from standard care and are considered responsible for recurrence of metastatic disease [3]. We propose that including ketosis and HBOT as a component of the standard treatment regimen would target a greater proportion of cancer cells simultaneously and thus provide a more comprehensive therapeutic response. A pre-operative period of metabolic therapy could shrink tumors and reduce infiltrative spread into nearby tissues, increasing the likelihood of successful and complete surgical resection of tumors. The anti-inflammatory effects of ketosis could replace the need for perioperative corticosteroids, which are prescribed for some patients to 165

183 reduce edema caused by surgery, but will also increase blood glucose levels and likely promote tumor growth [5, 6]. While our proposed metabolic therapy would likely kill some fraction of a patient s tumor burden on its own, it would also put significant physiological stress on any surviving tumor or circulating cancer cells. This metabolic stress could potentially sensitize the remaining cells to the effects of standard care. Indeed, studies have suggested a unique synergistic response between metabolic therapies and standard care. Scheck and colleagues reported complete remission in 82% of mice with glioma by pairing the ketogenic diet with radiation while no occurrences of remission were observed in the mice receiving either therapy alone [7]. Other studies have reported similar synergy between HBOT and chemotherapy or radiation [8-11]. Furthermore, synergy with metabolic therapy could allow for the use of smaller doses of cytotoxic agents, which could greatly improve patient quality of life. Also, inducing a state of ketosis during radiation, chemotherapy, or targeted drug treatment could protect healthy tissue from the damaging effects of these therapies [12]. Inclusion of other relatively non-toxic metabolic drugs that target cancer metabolism, like the glutamine-binding phenylbutyrate, the glucose-lowering metformin, or the fermentation-inhibiting dichloroacetate, could further enhance therapeutic response. Once in clinical remission, continuation of ketone supplementation and/or the KD, or inducing intermittent periods of ketosis, could maintain stress on any residual cancer cells and extend the period of remission temporarily or permanently. This novel combination of ketosis and HBO targets the dysregulated metabolic pathways that play vital roles in every step of cancer progression. The significant response reported here in the highly aggressive VM-M3 model is encouraging and strongly suggests that this therapy could be effective against metastatic tumors which are notoriously difficult to treat. In order to maximize therapeutic response, it will be important to test the effects of ketosis and HBOT as 166

184 part of a well-planned and comprehensive therapeutic regimen which will likely consist of a variety of metabolic therapies coupled to standard care. It is also possible that metabolic therapy could provide an effective, front-line treatment regimen for certain cancer types. Thus, it is critical that these cost-effective, readily implementable, non-toxic therapies are tested in a variety of preclinical and clinical settings to determine the extent of their therapeutic potential. We propose that combination ketosis and HBO metabolic therapy could offer novel therapeutic options for, and improve the outcome of, many patients including those with aggressive or latestage cancer. 6.2 References for Chapter Harrison LB, Shasha D, Homel P: Prevalence of anemia in cancer patients undergoing radiotherapy: prognostic significance and treatment. Oncology 2002, 63 Suppl 2: Dhup S, Dadhich RK, Porporato PE, Sonveaux P: Multiple biological activities of lactic acid in cancer: influences on tumor growth, angiogenesis and metastasis. Curr Pharm Des 2012, 18(10): Menendez JA, Joven J, Cufi S, Corominas-Faja B, Oliveras-Ferraros C, Cuyas E, Martin- Castillo B, Lopez-Bonet E, Alarcon T, Vazquez-Martin A: The Warburg effect version 2.0: metabolic reprogramming of cancer stem cells. Cell Cycle 2013, 12(8): Kroemer G, Pouyssegur J: Tumor cell metabolism: cancer's Achilles' heel. Cancer Cell 2008, 13(6): Seyfried T, Mukherjee P: Targeting energy metabolism in brain cancer: review and hypothesis. Nutr Metab (Lond) 2005, 2(d2af0c96-bf3d-ee23-f c210fdc5): Seyfried TN, Shelton LM, Mukherjee P: Does the existing standard of care increase glioblastoma energy metabolism? Lancet Oncol 2010, 11(9): Abdelwahab M, Fenton K, Preul M, Rho J, Lynch A, Stafford P, Scheck A: The ketogenic diet is an effective adjuvant to radiation therapy for the treatment of malignant glioma. PLoS One 2012, Kalns J, Krock L, Piepmeier E, Jr.: The effect of hyperbaric oxygen on growth and chemosensitivity of metastatic prostate cancer. Anticancer research 1998, 18(1A): Petre P, Baciewicz F, Tigan S, Spears J: Hyperbaric oxygen as a chemotherapy adjuvant in the treatment of metastatic lung tumors in a rat model. The Journal of thoracic and cardiovascular surgery 2003, 125(1):

185 10. Stuhr L, Iversen V, Straume O, Maehle B, Reed R: Hyperbaric oxygen alone or combined with 5-FU attenuates growth of DMBA-induced rat mammary tumors. Cancer letters 2004, 210: Kohshi K, Kinoshita Y, Imada H, Kunugita N, Abe H, Terashima H, Tokui N, Uemura S: Effects of radiotherapy after hyperbaric oxygenation on malignant gliomas. Br J Cancer 1999, 80(1-2): Maalouf M, Rho J, Mattson M: The neuroprotective properties of calorie restriction, the ketogenic diet, and ketone bodies. Brain research reviews 2009, 59(2):

186 APPENDIX A: MATERIALS AND METHODS A.1 Ethics Statement All animal studies were approved by and preformed within strict adherence to the University of South Florida s Institutional Animal Care and Use Committee (IACUC) protocols R4137 and R A.2 Materials and Methods for Included Manuscripts A.2.1 Materials and methods pertaining to the following experiments are described in Poff, et al, The Ketogenic Diet and Hyperbaric Oxygen Therapy Prolong Survival in Mice with Systemic Metastatic Cancer, PLOS One, See Appendix C. VM/dk mouse husbandry Cell culture VM-M3/Fluc inoculation for tumor implantation Diet therapy Hyperbaric oxygen therapy Blood glucose and ketone measurements Weight measurements Ex Vivo bioluminescent imaging and tumor growth analysis 169

187 Survival analysis Statistics A.2.2 Materials and methods pertaining to the following experiments are described in Poff, et al, Ketone supplementation decreases tumor cell viability and prolongs survival of mice with metastatic cancer, Int. J. of Cancer, See Appendix C. Cell culture VM-M3 proliferation VM-M3 Viability VM/dk mouse husbandry VM-M3/Fluc inoculation for tumor implantation Ketone supplementation and diet administration Blood glucose and ketone measurements Weight measurements In Vivo bioluminescent imaging and tumor growth analysis Survival analysis Statistics A.2.3 Materials and methods pertaining to the following experiments are described in Poff, et al, Non-toxic metabolic management of metastatic cancer in VM mice: Novel combination of ketogenic diet, ketone supplementation, and hyperbaric oxygen therapy, (Accepted with revisions, PLOS One). See Appendix C. 170

188 Cell culture VM/dk mouse husbandry VM-M3/Fluc inoculation for tumor implantation Dietary administration Hyperbaric oxygen therapy Blood glucose and ketone measurements Weight measurements In Vivo bioluminescent imaging and tumor growth analysis Survival analysis VM-M3 cell proliferation VM-M3 cell viability VM-M3 cell ROS detection Western blot analysis Statistics A.3 Materials and Methods for Unpublished Supporting Data A.3.1 Western Blot Analysis of pakt (Figures 3.8, 4.2) VM-M3 cells were grown for 7 days in control media with or without 5mM βhb or daily HBOT (100% O2, 2.5 ATA, 90 min). Media was replaced every 48 hours during the treatment period. Cells were collected by scraping and protein was extracted with sonication in HEPES lysis buffer containing protease and phosphatase inhibitors. Protein isolate was separated on a 4-20% Criterion Gel (Bio-Rad) and electrotransferred onto a nitrocellulose membrane. 171

189 Membranes were blocked with 5% non-fat dry milk and immunoblotted with anti-phospho-akt (Ser473) rabbit monoclonal antibody (1:1000, Cell Signaling #4060S) and HRP-conjugated goat anti-rabbit secondary antibody. Membranes were also probed with anti-pan-actin rabbit polyclonal antibody (1:2000; Cell Signaling #4968S) as a loading control. Bands were imaged on film using a standard chemiluminescent visualization protocol and band density was determined with Image J software (NIH). Statistical analysis was performed using unpaired t- tests. Results were considered significant when p<0.05. A.3.2 Histological Analysis of Tumor Morphology and Immunohistochemical Analysis of Tumor Vascularization (Figures 3.7, 4.11, 5.7) Approximately 10 6 VM-M3/Fluc cells in 300 µl sterile PBS were subcutaneously implanted into the abdomen of 2-4 month old male VM/dk mice. On the day of inoculation, mice were randomly assigned to either control or treatment groups (N=8). Control animals were fed standard rodent chow (2018 Teklad Global 18% Protein Rodent Diet, Harlan) ad libitum. Treated animals received KD-USF (Harlan Laboratories), a custom- designed ketogenic diet, with 10% KE mixed in by weight, fed ad libitum, and HBOT sessions (100% O2, 2.5 ATA, 90 min) three times per week. The macronutrient profile and caloric density of SD, KD-USF, and KE are shown in Table 2. After 21 days, animals were humanely euthanized and livers were harvested and preserved in 10% neutral, phosphate buffered formalin. The liver tissue was paraffin-embedded, cut into 5 micron sections, and mounted onto microscope slides at the USF Morsani College of Medicine Histology Core. One set of slides was stained with hematoxylin and eosin and mounted with cover slips for morphological analysis. Another set of slides was probed for the endothelial cell marker CD31 to visualize blood vessels. These slides were 172

190 rehydrated through a series of xylene and ethanol washes, and antigen was retrieved by a standard heat-mediated citrate buffer method. Slides were incubated in 0.3% H2O2 in dh2o to inhibit endogenous peroxide activity. Immunohistochemical analysis of the slides was performed using the VECTASTAIN Elite ABC kit. Slides were blocked for 30 minutes in normal blocking serum, and then incubated in anti-cd31 antibody (1:25; ab28364, Abcam) for one hour at room temperature. Slides were then incubated in biotinylated anti-rabbit secondary antibody in normal blocking serum for 30 minutes at room temperature, followed by VECTASTAIN ABC reagent containing avidin and biotinylated horseradish peroxidase solution for 30 minutes at room temperature. Slides were developed using the VECTOR NovaRED Peroxidase (HRP) Substrate Kit. Slides were counterstained with hematoxylin, washed, mounted with coverslips, and visualized with light microscopy. At least 3 frames containing tumor tissue per sample were analyzed. The number of CD31 + blood vessels associated with tumors in each frame was counted and compared between control and treated groups as a measure of tumor vascularization. Statistical analysis was performed with unpaired t-test. Results were considered significant when p<0.05. Table A.1. Macronutrient profile and caloric density of diets and ketone supplements. Macronutrient Information Standard Diet (SD) Ketovolve (KD) KD-USF (KD2) 1,3- Butanediol (BD) Ketone Ester (KE) % Cal from Fat N/A N/A % Cal from Protein N/A N/A % Cal from Carbohydrate N/A N/A Caloric Density 3.1 Kcal/g 7.12 Kcal/g 4.7 Kcal/g 5.58 Kcal/g 6.0 Kcal/g 173

191 A.3.3 Chronic Oral Administration of Ketone Supplements in Rats (Fig , Table 1) A Intragastric Gavage to Induce Ketosis with BD and KE Male Sprague-Dawley rats ( g) were purchased from Harlan Laboratories and shipped directly to the USF Morsani College of Medicine Vivarium for this study. Rats received a daily 5g/kg dose of either water (control), BD, or KE via intragastric gavage for 29 days (N=8). Gavage was performed at the same time daily, and rats were weighed weekly to ensure proper dosing. A Blood Glucose, Ketone, and Lipid Measurements (Figures ) On the first day (day 0), and every 7 days for the duration of the study (days 7, 14, 21, 28), blood glucose and βhb were measured over the 8 hours following water or ketone supplement administration. Animals were fasted for 4 hours (water available) prior to intragastric gavage to stabilize blood metabolites. Immediately prior to (baseline), and 0.5, 1, 4, and 8 hours following intragastric gavage, whole blood samples (approximately 10 µl) were taken from the saphenous vein to measure blood glucose and βhb with the Precision Xtra Blood Glucose & Ketone Monitoring System (Abbott Laboratories). On day 0 and 28, baseline blood cholesterol, high density lipoprotein (HDL), and triglycerides were measured with the CardioChek blood lipid analyzer (Polymer Technology Systems, Inc.). Low density lipoprotein (LDL) levels were derived using the Friedewald equation: LDL = Total Cholesterol HDL (Triglyceride/5) [1, 2]. Statistical analysis was performed with one-way ANOVA. Results were considered significant when p<

192 A Metabolomics Profiling of KE Treated Rats (Table 4.1) On day 29, approximately 500 µl of whole blood was collected from the saphenous vein of control and KE-treated rats 4 hours after intragastric gavage. Serum was separated by centrifugation using Microtainer Tubes with Serum Separator (Becton Dickinson). Serum was transferred to screw-top cryovials and flash frozen in liquid N2. Frozen serum samples were shipped to Metabolon, Inc. for global metabolomics profiling using liquid chromatographytandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS). The concentration of 388 named biochemical of known identity were analyzed in the serum samples. Welch s two-sample t-test was used to determine which metabolites differed significantly in concentration between control and KE-treated animals. Results were considered significant when p<0.05. A.3.4 Ketone-Supplemented Ketogenic Diets Survival Study (Figures ) Approximately 1 million VM-M3/Fluc cells were implanted s.c. into the abdomen of 2-4 month old male VM/dk mice. On the day of inoculation, mice were randomly assigned to either control, KD+BD, or KD+KE (N 7). Control mice were fed standard rodent chow (2018 Teklad Global 18% Protein Rodent Diet, Harlan) ad libitum. KD+BD-treated mice were fed KD-USF mixed with 20% BD and 1% saccharin (for palatability) by weight ad libitum. KD+KE-treated animals were fed KD-USF mixed with 10% KE and 1% saccharin (for palatability) by weight ad libitum. The macronutrient profile and caloric density of SD, KD-USF, BD, KE are provided in Table A.1. The methods regarding blood glucose and ketones, weight, tumor growth, metastatic spread, and survival analysis were the same as previously described (Chapters 3, 4) [3, 4]. 175

193 A.3.4 Metastatic Spread - Ex Vivo Organ Bioluminescence (Figures 4.10 and 5.6) Approximately 1 million VM-M3/Fluc cells implanted s.c. into the abdomen of 8-16 week old male VM/dk mice. On the day of inoculation, mice were randomly assigned to either control or treatment groups (N=8). Control mice were fed standard rodent chow (2018 Teklad Global 18% Protein Rodent Diet, Harlan) ad libitum. KD+KE and KD+KE+HBOT-treated animals received KD-USF with 10% KE and 1% saccharin by weight ad libitum. KD+KE+HBOT mice also received HBOT (100% O2, 2.5 ATA, 90 min) three times weekly. The macronutrient profile and caloric density of SD, KD-USF, and KE are provided in Table A.1. After 3 weeks, animals were humanely euthanized, and brain, lungs, liver, kidneys, spleen, and a sample of adipose tissue were harvested. The organs were immediately placed in a petri dish containing 300 µg/ml sterile PBS for 5 min. Ex vivo organ bioluminescence was then measured using the IVIS Lumina cooled CCD camera system and Living Image software (Caliper LS). Bioluminescent signal (photons/sec) of each organ was used to compare extent of metastatic spread between control and treated animals. Statistical analysis was performed with unpaired t-test. Results were considered significant when p<0.05. A.4 References for Appendix A 1. Warnick GR, Knopp RH, Fitzpatrick V, Branson L: Estimating low-density lipoprotein cholesterol by the Friedewald equation is adequate for classifying patients on the basis of nationally recommended cutpoints. Clinical chemistry 1990, 36(1): Friedewald WT, Levy RI, Fredrickson DS: Estimation of the concentration of lowdensity lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clinical chemistry 1972, 18(6): Poff AM, Ari C, Seyfried TN, D'Agostino DP: The ketogenic diet and hyperbaric oxygen therapy prolong survival in mice with systemic metastatic cancer. PLoS One 2013, 8(6):e

194 4. Poff AM, Ari C, Arnold P, Seyfried TN, D'Agostino DP: Ketone supplementation decreases tumor cell viability and prolongs survival of mice with metastatic cancer. Int J Cancer

195 APPENDIX B: COPYRIGHT PERMISSIONS B.1 Copyright Permissions for: Poff A, Ari C, Seyfried T, D Agostino D. (2013) The Ketogenic Diet and Hyperbaric Oxygen Therapy Prolong Survival in Mice with Systemic Metastatic Cancer. PLOS One Permission to Include Published Work Citation: Poff AM, Ari C, Seyfried TN, D Agostino DP (2013) The Ketogenic Diet and Hyperbaric Oxygen Therapy Prolong Survival in Mice with Systemic Metastatic Cancer. PLoS ONE 8(6): e doi: /journal.pone Open-Access License No Permission Required CCAL 3.0 License PLOS applies the Creative Commons Attribution (CC BY) license to all works we publish (read the human-readable summary or the full license legal code). Under the CC BY license, authors retain ownership of the copyright for their article, but authors allow anyone to download, reuse, reprint, modify, distribute, and/or copy articles in PLOS journals, so long as the original authors and source are cited. No permission is required from the authors or the publishers. In most cases, appropriate attribution can be provided by simply citing the original article (e.g., Kaltenbach LS et al. (2007) Huntingtin Interacting Proteins Are Genetic Modifiers of Neurodegeneration. PLOS Genet 3(5): e82. doi: /journal.pgen ). This broad license was developed to facilitate open access to, and free use of, original works of all types. Applying this standard license to your own work will ensure your right to make your work freely and openly available. Learn more about open access. For queries about the license, please contact us

196 B.2 Copyright Permissions for: Poff A, Ari C, Arnold P, Seyfried T, D Agostino D. (2014) Ketone Supplementation Decreases Tumor Cell Viability and Prolongs Survival of Mice with Metastatic Cancer. Int. J. of Cancer Permission for inclusion of the aforementioned publication was granted via (see below). The copyright notice is provided on the following page. Dear Angela: Thank you for your request. Permission is hereby granted for the use requested subject to the usual acknowledgements (author, title of material, title of book/journal, ourselves as publisher). You shall also duplicate the copyright notice that appears in the Wiley publication in your use of the Material. Any third party material is expressly excluded from this permission. If any of the material you wish to use appears within our work with credit to another source, authorization from that source must be obtained. This permission does not include the right to grant others permission to photocopy or otherwise reproduce this material except for accessible versions made by non-profit organizations serving the blind, visually impaired and other persons with print disabilities (VIPs). Sincerely, Paulette Goldweber Associate Manager, Permissions/Global Rights pgoldweb@wiley.com permissions@wiley.com John Wiley & Sons, Inc. 111 River Street Hoboken, NJ

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204 APPENDIX C: PUBLISHED MANUSCRIPTS This appendix contains the original publications for the following references: Poff A, Ari C, Seyfried T, D Agostino D. (2013) The Ketogenic Diet and Hyperbaric Oxygen Therapy Prolong Survival in Mice with Systemic Metastatic Cancer. PLOS One. Poff A, Ari C, Arnold P, Seyfried T, D Agostino D. (2014) Ketone Supplementation Decreases Tumor Cell Viability and Prolongs Survival of Mice with Metastatic Cancer. Int. J. of Cancer. Poff A, Ward N, Seyfried T, Arnold P, D Agostino D. (2014) Non-toxic metabolic management of metastatic cancer in VM mice: Novel combination of ketogenic diet, ketone supplementation, and hyperbaric oxygen therapy. Manuscript accepted with revisions: PLOS One. 187

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246 ABOUT THE AUTHOR Angela Poff was born in Fort Smith, AR and raised in Greenwood, AR. She attended Hendrix College in Conway, AR where she received a B.A. in Biochemistry and Molecular Biology. In 2010, Angela moved to Tampa, FL to pursue her graduate training as a student in the Ph.D. Program in Integrated Biomedical Sciences at the University of South Florida. Angela performed her dissertation research in the Hyperbaric Biomedical Research Laboratory of the Department of Molecular Pharmacology and Physiology under the mentorship of Dr. Dominic D Agostino. During her time at USF, she presented her research at many national and international conferences. She served on the executive board of the Association of Medical Sciences Graduate Students from , serving as president from Angela received her Masters of Science in Medical Science in 2013 and successfully defended her dissertation on June 30, 2014.