S6K1 mediates oncogenic glycolysis in Pten deficient leukemia A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Ph.D.) in the Department of Cancer and Cell Biology of the College of Medicine by Preeti Tandon M.S. Bowling Green State University, 2004 ABSTRACT Hyperactive Akt signaling triggers glycolysis and apoptosis resistance in human cancer. Because sustained glycolysis is required for Akt dependent apoptosis resistance, we investigated the downstream signaling components that mediate Akt dependent increases in glycolysis in cells deficient for Pten, a negative regulator of the PI3K/Akt pathway. Genetic inactivation of the ribosomal protein S6 Kinase 1 (S6K1) in Pten-deficient cells prevented glycolysis, triggered Bax translocation and committed cells to apoptosis. Pharmacological S6K1 inhibition using a small molecule kinase inhibitor recapitulated the effects of genetically inactivating S6K1. Inactivation of S6K1 was associated with decreased expression of the pro-glycolytic HIF1α transcription factor. Restoring HIF1α expression was sufficient to restore both glycolysis and cell survival in S6K1-deficient cells. Conversely, inhibiting HIF1α expression in Pten deficient cells resulted in decreased glycolysis and cell survival, mimicking the loss of S6K1. In vivo, S6K1 deficiency delayed the development of lethal disease in a Pten deficient mouse model of leukemia. Thus, together the data suggest that S6K1 is a useful target for counteracting the metabolic program that supports apoptosis resistance in Pten-deficient cancers. iii iv ACKNOWLEDGEMENTS I would first and foremost like to thank Dr David Plas for being my mentor. Your guidance and advice have been instrumental in my progress and development as a scientist. You have been a great teacher and I have learnt a lot from you over the years. Thank you for everything- all the impromptu quizzes during lab meetings, making sure that we always did the best experiment and for encouraging us to never say no to an opportunity to present. You have been an excellent role model and the best advisor that anyone could ever ask for. I would like to thank members of my thesis committee – Dr. George Thomas, Dr. James Mulloy, Dr. Angela Drew and Dr. Maria Czyzyk Krzeska for their support, encouragement and critical review of this research. I would also like to thank all of the past and present members of the Plas lab for making it an enjoyable experience. Thank you all for your technical assistance, valuable scientific discussions and most importantly your friendship and support. I especially want to thank Shikha for being a great friend and a wonderful co-worker. A special thanks to my parents for their unwavering support and love. Thank you for inspiring me to be the best at whatever I do. Thanks mom for the unlimited supply of scrumptious food that you sent my way that allowed me to utilize those kitchen hours in the lab to do more experiments. Thank you papa for never letting me give up. Thanks to my parents-in-law for their support and well wishes. I would also like to thank my sister, brother-in-law and their beautiful kids- Saahil and Saaz. Their smiling faces made me forget the disappointment of failed experiments. v Lastly but most importantly, I would like to thank my husband, Ritesh, for his unconditional love and support. Thank you for believing in me when I found it difficult to believe in myself. I couldn’t have done this without your constant encouragement, patience, love and strength. vi Table of Contents List of Figures………………………………………………………………………….9 Chapter I: Introduction 11 References………………………………………………………………………………41 Figures…………………………………………………………………………………..53 Chapter II: Requirement for ribosomal protein S6 Kinase 1 to mediate 57 glycolysis and apoptosis resistance induced by Pten deficiency Abstract………………………………………………………………………………….58 Introduction……………………………………………………………………………...59 Results…………………………………………………………………………………... 61 Discussion………………………………………………………………………………..68 Materials and Methods…………………………………………………………………..70 References………………………………………………………………………………..75 Figures……………………………………………………………………………………78 Chapter III: Analysis of an S6K1 inhibitor for counteracting glycolysis and 89 survival in Pten deficient cells Abstract……………………………………………………………………………………90 Introduction………………………………………………………………………………..91 Results……………………………………………………………………………………..93 Discussion………………………………………………………………………………….95 Materials and Methods…………………………………………………………………….96 References………………………………………………………………………………….98 Figures………………………………………………………………………………………99 Chapter IV: Conclusions and Discussion 102 References…………………………………………………………………………………..117 Figures………………………………………………………………………………………120 8 List of Figures Chapter I Figure 1……………………………………………………………………………………53 Figure 2……………………………………………………………………………………54 Figure 3…………………………………………………………………………………….55 Figure 4…………………………………………………………………………………….56 Chapter II Figure 1…………………………………………………………………………………….78 Figure 2…………………………………………………………………………………….79 Figure 3…………………………………………………………………………………….80 Figure 4……………………………………………………………………………………..81 Figure 5……………………………………………………………………………………..82 Supp. Figure 1………………………………………………………………………………83 Supp. Figure 2………………………………………………………………………………84 Supp. Figure 3………………………………………………………………………………85 Supp. Figure 4………………………………………………………………………………86 Supp. Figure 5……………………………………………………………………………....87 Supp. Figure 6………………………………………………………………………………88 Chapter III Figure 1………………………………………………………………………………………99 Figure 2………………………………………………………………………………………100 Figure 3……………………………………………………………………………………....101 9 Chapter IV Figure 1………………………………………………………………………………………120 Figure 2………………………………………………………………………………………121 Figure 3……………………………………………………………………………………....122 Figure 4……………………………………………………………………………………....123 Figure 5……………………………………………………………………………………….124 Figure 6……………………………………………………………………………………….125 Figure 7……………………………………………………………………………………….126 10 CHAPTER I Introduction 11 Cancer Cell Metabolism: The Warburg Effect Dysregulated cellular metabolism is a distinguishing feature of transformed cells. More than 80 years ago, Otto Warburg showed that tumor cells metabolize glucose to lactate at a much higher rate than normal cells despite the presence of adequate oxygen- a phenomenon now known as the Warburg Effect or aerobic glycolysis (1). This effect has since been observed in different tumor types and is considered an essential feature of cancer cells. Although the Warburg effect is a hallmark of cancer, its regulatory mechanism remains obscure. Identifying factors that regulate cancer cell metabolism will enhance our understanding of cancer development and progression and provide novel approaches for cancer therapy. A key question in cancer biology is why cancer cells preferentially activate glycolysis, which yields only 2 ATP per molecule of glucose, instead of glucose oxidation, which yields up to 36 ATP. Warburg reasoned that defects in mitochondrial respiration cause increased aerobic glycolysis in cancer cells (2). The discovery of oncogenic mutations in oxidative phosphorylation (OXPHOS) genes, such as, fumarate hydratase (FH), succinate dehydrogenase (SDH) and isocitrate dehydrogenase (IDH1 and IDH2) validated Warburg’s hypothesis (3-5). However, these mutations are rare and only occur in a small subset of cancers (6). The majority of tumors retain the ability to consume oxygen at rates comparable to normal cells, suggesting other mechanisms underlying the metabolic reprogramming seen in tumor cells (7). An alternative explanation is that glycolysis confers proliferative advantages upon cancer cells. In addition to requiring ATP, cancer cells also require building blocks such as nucleotides, lipids, proteins and fatty acids to sustain uncontrolled cell proliferation. By preventing glucose oxidation via OXPHOS for maximal ATP production, cancer cells can divert glucose carbons 12 into macromolecular precursors such as acetyl co-A for fatty acid synthesis, ribose for nucleotide synthesis and 3-phosphoglycerate (3-PG) for amino acid synthesis (8-10). These changes in metabolic destinations of glucose can be regulated by altered expression of different isoforms of the glycolytic enzyme, pyruvate kinase (PK). Pyruvate kinase catalyzes the penultimate step in glycolysis, the conversion of phosphoenolpyruvate (PEP) to pyruvate. Cancer cells preferentially express pyruvate kinase M2 (PKM2) in place of PKM1, which is expressed in non-proliferating adult tissues (11). The PKM2 isoform is less active in converting PEP to pyruvate and generating ATP than PKM1 (11). PKM2 expression triggers the accumulation of the upstream metabolite PEP, which can now be incorporated into biosynthetic processes to support cell proliferation. Furthermore, PEP can donate phosphate via a phosphotransferase reaction to the metabolic enzyme phosphoglycerate mutase1 (PGAM1), producing phospho- PGAM1 and pyruvate (12). Phosphorylation of PGAM1 increases the mutase function of the enzyme, triggering a positive feedback loop whereby PEP increases the activity of upstream glycolytic pathway enzymes. Thus, pyruvate production by this alternative pathway may provide an additional mechanism to promote the redistribution of metabolites upstream of PGAM1 into biosynthetic pathways. Another survival advantage of switching from oxidative to aerobic metabolism is decreased production of reactive oxygen species (ROS). Mitochondrial respiration is the major source of ROS production in cells. Excessive