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Nuclear pyruvate kinase M2 functional study in cancer cells

Dissertation
Author: Xueliang Gao
Abstract:
Cancer cells take more glucose to provide energy and phosphoryl intermediates for cancer progression. Meanwhile, energy-provider function of mitochondria in cancer cells is disrupted. This phenomenon is so-called Warburg effect, which is discovered over eighty years ago. The detail mechanisms for Warburg effect are not well defined. How glycolytic enzymes contribute to cancer progression is not well known. PKM2 is a glycolytic enzyme dominantly localized in the cytosol, catalyzing the production of ATP from PEP. In this study, we discovered that there were more nuclear PKM2 expressed in highly proliferative cancer cells. The nuclear PKM2 levels are correlated with cell proliferation rates. According to our microarray analyses, MEK5 gene was upregulated in PKM2 overexpression cells. Our studies showed that PKM2 regulated MEK5 gene transcription to promote cell proliferation. Moreover, nuclear PKM2 phosphorylated Stat3 at Y705 site using PEP as a phosphoryl group donor to regulate MEK5 gene transcription. Our study also showed that double phosphorylated p68 RNA helicase at Y593/595 interacted with PKM2 at its FBP binding site. Under the stimulation of growth factors, p68 interacted with PKM2 to promote the conversion from tetrameric to dimeric form so as to regulate its protein kinase activity. Overexpression PKM2 in less aggressive cancer cells induced the formation of multinuclei by regulating Cdc14A gene transcription. Overall, this study presents a step forward in understanding the Warburg effect. Index words. PKM2, P68 RNA helicase, MEK5, Stat3, Cdc14A, Tyrosine phosphorylation, Gene transcription, Cell cycle, Glycolysis.

v TABLE OF CONTENTS

ACKNOWLEDGEMENTS iv

LIST OF TABLES xiv

LIST OF FIGURES xv

CHAPTER 1 GENERAL INTRODUCITON 1

1.1 Cancer metabolism 1

1.1.1 Tumors and Cancer 1

1.1.2 Cancer Metabolism and Warburg Effect 3

1.2 Possible Causes of Metabolic Changes in Cancer Cells 4

1.2.1 Tumor Microenvironment Affects Tumor Metabolism 4

1.2.2 Oncogenes and Tumor Suppressor Genes Regulate Cancer Cell 6

Metabolism

1.2.3 Mitochondrial Malfunction in Cancer Cells 9

1.2.4 Metabolic Malfunction is a Potential Cancer Therapy Method 11

1.3 The Introduction of Glycolysis---Functional and Regulational 13

Mechanisms of Glycolysis in Cancer Cells

1.4 Pyruvate Kinase Type M2 16

1.5 p68 RNA Helicase 21

1.6 MEK5 and MAP Kinase Pathway 25

1.6.1 MAPK Family Members and Their Biological Functions 25

1.6.2 ERK5-MEK5 Signal Pathway 28

1.7 Stat Family and The Biological Function of Stat3 30

1.8 Cdc14 Regulates Mitosis Exit in Eukaryotic Cells 35

vi 1.9 Aims of the Dissertation 38

1.10 References 39

CHAPTER 2 PYRUVATE KINASE M2 PROTES CELL PROLIFERATION 51

BY REGULATING GENE TRANSCRIPTION

2.1 Abstract 51

2.2 Introduction 51

2.3 Results 53

2.3.1 Nuclear PKM2 Levels Correlate with Tumor Progression and 53

Expression of PKM2 Promotes Cell Proliferation

2.3.2 PKM2 Activates Transcription of MEK5 55

2.3.3 PKM2 Upregulates MEK5 Transcription by Activation of Stat3 58

2.3.4 Expression of PKM2 did not Alter Production of Pyruvate and 61

Lactate

2.4 Discussion 62

2.5 Materials and Methods 64

2.5.1 Reagents, Cell lines, and Antibodies 64

2.5.2 RNA Interference, Subcellular Extracts Preparation, Co- 65

immunoprecipitation, immunoblot, Cell Proliferation Assays,

and Chromatin immunoprecipitation (ChIP)

2.5.3 Expression of PKM2 by Adenovirus Using Commercial AdEasy 65

System

2.5.4 Cellular Pyruvate and Lactate 66

2.5.5 Microarray Analyses of Gene Expressions 66

vii 2.5.6 Real Time PCR and RT-PCR 67

2.5.7 Gel-Mobility Shift and Super-Shift Assays 68

2.5.8 ChIP, Re-ChIP 68

2.6 References 69

CHAPTER 3 NUCLEAR PYRUVATE KINASE M2 IS A PROTEIN KINASE 119

3.1 Abstract 119

3.2 Introduction 119

3.3 Results 120

3.3.1 PKM2 is a Protein Kinase 120

3.3.2 Dimeric PKM2 is the Active Protein Kinase 121

3.3.3 Binding Tyrosyl Phosphor-protein at the FBP Site Converts the 124

Tetramer PKM2 to the Dimmer and Reciprocally Regulates the

Protein Kinase and Pyruvate Kinase Activities

3.3.4 PKM2 Kinase Substrates Bind to the ADP Binding Site for 129

Phosphorylation

3.3.5 PKM2 Protein Kinase Substrates Bind to the ADP Binding Site 130

3.3.6 Expression of R399E Increased Stat3 Phosphorylation in Cells 132

and Promoted Cell Proliferation

3.3.7 Growth Factor Stimulations Result in Increase in Dimeric PKM2 133

in Cells

3.4 Discussion 134

3.5 Materials and Methods 136

3.5.1 Reagents, Cell lines, and Antibodies 136

viii 3.5.2 Plasmids Construction 136

3.5.3 Expression and Purification of Recombinant PKM2 137

3.5.4 Transfection and RNA Interference, Subcellular Extracts 137

Preparation, Immunoprecipitation and Immuno Blots, and PCR

and RT-PCR

3.5.5 Size-exclusion Chromatography and Non-denaturing Gel 138

Electrophoresis

3.5.6 PKM2 and Peptide Interaction 139

3.5.7 In vitro Protein Kinase and Pyruvate Kinase Assays 139

3.5.8 Peptide Pull Down and Protein Identification by Mass 140

Spectroscopy

3.6 References 141

CHAPTER 4 NUCLEAR PKM2 REGULATES CDC14A EXPRESSION TO 161

INDUCE MULTINUCLEUS

4.1 Abstract 161

4.2 Introduction 161

4.3 Results 164

4.3.1 Exogenous Overexpression of PKM2 and p68 RNA Helicase 164

Induces Multi-nuclei in Cancer Cells

4.3.2 Nuclear PKM2 Regulates the Expression of Cdc14A 166

4.3.3 PKM2 and p68 RNA Helicase Co-regulate Cdc14A Protein 166

Expression

4.3.4 PKM2 and p68 RNA Helicase Bind to Cdc14A Promoter Region 168

ix 4.3.5 Cdc14A Mediates the Effect of PKM2 in Induction of Formation 168

of Multinuclei

4.3.6 PKM2 Overexpression Induces formation of Multi-nucleus Only 169

in Less Aggressive Cancer Cells

4.4 Discussion 170

4.5 Materials and Methods 172

4.5.1 Plasmids Construction, Reagents and Antibodies 172

4.5.2 Cell culture, Transient Transfection Assays 172

4.5.3 Relative Real Time PCR (RT-PCR) 173

4.5.4 Chromatin Immunoprecipitation 173

4.5.5 RNA Interference 174

4.5.6 Subcellular Extracts Preparation, Co-immunoprecipitation and 174

Western blot

4.5.7 Immunostaining and Multinucleated Cells Analysis 175

4.6 References 175

CHAPTER 5 CONCLUSIONS AND DISCUSSIONS 192

5.1 Conclusions 192

5.2 Nuclear PKM2 Interacts with Tyrosine-phosphorylated p68 RNA 192

Helicase

5.3 Tyrosine-phosphorylated p68 Peptides Change the Quaternary 193

Structure of PKM2

5.4 Nuclear PKM2 Regulates MEK5 Gene Transcription to Promote Cell 195

Proliferation

x 5.5 Nuclear PKM2 Phosphorylates Stat3 in the Nucleus to Regulate Its 196

Transcriptional Activity

5.6 Overexpression PKM2 Forms Multinucleus in Less aggressive Cancer 197

Cells

5.7 Dimeric PKM2 Phosphorylates Stat3 Using PEP as Phosphoryl Group 198

Donor

5.8 Arginine 399 Changes the Quaternary Structure of PKM2 199

5.9 References 199

CHAPTER 6 MATERIALS AND METHODS 201

6.1 Nucleic Acids Related Techniques 201

6.1.1 Mini Preparation for DNA 201

6.1.2 Midi Preparation for DNA 202

6.1.3 Agarose Gel Electrophoresis and Gel Extraction of DNA 203

6.1.4 Quantification of DNA and RNA 203

6.1.5 Polymerase Chain Reaction (PCR) Method 204

6.1.6 Restriction Enzyme Digestion and Plasmids Construction 204

6.1.7 Site-directed Mutation Method 205

6.1.8 DNA Sequencing 206

6.1.9 Ethanol Purification of DNA 206

6.1.10 RNA Extraction 206

6.1.11 Reverse Transcription PCR 207

6.1.12 Relative Real Time PCR 208

6.1.13 Chromatin Immunoprecipitation 208

xi 6.1.14 ChIP-on-chip 209

6.1.15 Microarray Analysis 210

6.1.16 Electrophoretic Mobility Shift Assay 210

6.2 Bacterial Techniques 211

6.2.1 Bacterial Culture and Storage 211

6.2.2 Transformation 211

6.3 Protein Related Techniques 212

6.3.1 Recombinant PKM2 Protein Expression and Purification 212

6.3.2 Gel Filtration 213

6.3.3 Protein Quantification 214

6.3.4 Sodium Dodecyl Sulfate Polyacryalmide Gel Electrophoresis 214

(SDS-PAGE)

6.3.5 Coomassie Blue Staining 216

6.3.6 GelCode Staining 216

6.3.7 Native Gel Preparation 217

6.3.8 Protein In-gel Digestion 217

6.3.9 Protein Identification by Peptide Mass Fingerprinting 218

6.3.10 Antibody Generation and Purification 218

6.3.11 Kinase Assay 219

6.4 Mammalian Cell Techniques 220

6.4.1 Mammalian Cell Culture and Storage 220

6.4.2 Transient Transfection Method 220

6.4.3 RNA Interference 221

xii 6.4.4 Growth Factor Treatment of Cells 222

6.4.5 Whole Cell Lysate Preparation 222

6.4.6 Nuclear Extract and Cytoplasmic Extract Preparation 223

6.4.7 Suspension Growth of HeLa S3 cell 223

6.4.8 Large Scale Nuclear Extract Preparation 224

6.4.9 Immunoprecipitation (IP) and Co-immunoprecipitation (Co-IP) 226

6.4.10 Western Blot 227

6.4.11 Cell Proliferation Assay 228

6.4.12 Recombinant Adenovirus Generation and Infection in 228

Mammalian Cells

6.4.13 Metabolism Measurement 230

6.5 Materials 231

CHAPTER 7 APPENDIX 239

7.1 Detection of Associated Nuclear Proteins to Phosphorylated p68 RNA 239

Helicase

7. 1.1 Abstract 239

7.1.2 Introduction 239

7.1.3 Methods 240

7.1.3.1 Co-Immunoprecipitation and Peptide Pulldown 240

7.1.3.2 Two-Dimensional SDS-PAGE Analysis 240

7.1.3.3 Protein Identification by Peptide Mass Fingerprinting 241

7.1.4 Results and Discussions 242

7.1.4.1 Identification of Phosphorylated Residues of p68 RNA 242

xiii Helicase with MALDI-TOF

7.1.4.2 Detection of p68 Associated Nuclear Proteins Using 2-D 243

gel

7.1.4.3 Analysis of Nuclear Associated Proteins to 244

Phosphorylated p68 Using Peptide-protein Interaction

Screening Method

7.1.5 References 244

xiv LIST OF TABLES Table 1 List for Gene Expressions Regulated by PKM2 Overexpression in 89

SW480 Cells

Table 2 Antibody List 231

Table 3 Primers List 233

Table 4 Mammalian Cell Lines 235

Table 5 Chemicals 236

Table 6 Experimental Kits 238

xv LIST OF FIGURES Figure 2.1 PKM2 Promotes Cell Proliferation 71

Figure 2.2 PKM2 Upregulates MEK5 Transcription 73

Figure 2.3 Expression of MEK5 Mediates the Effects of PKM2 in Cell 75

Proliferation

Figure 2.4 PKM2 Upregulates MEK5 Transcription via Activation of Stat3 77

Figure 2.5 PKM2 Upregulates MEK5 Transcription by Promoting Stat3 DNA 79

Interaction

Figure 2.6 PKM2 Phosphorylates Stat3 81

Figure 2.7 Expression PKM2 in Cancer Cells 83

Figure 2.8 PKM2 Interacts with Stat3 to Regulate MEK5 Gene Transcription 85

Figure 2.9 PKM2 is Associated with Stat3 Promoter Region 87

Figure 3.1 PKM2 Phosphorylates GST-stat3. 143

Figure 3.2 Dimeric PKM2 is Active Protein Kinase 145

Figure 3.3 Tyrosine Phosphor-peptide Converts Tetramer PKM2 to a Dimer 147

and Activates Protein Kinase Activity

Figure 3.4 The R399E Mutant Phosphorylates Stat3 in Cells and Expression 149

of the Mutant Promotes Cell Proliferation

Figure 3.5 Growth Factor Stimulation Leads to Tetramer to Dimer 151

Conversion of PKM2

Figure 3.6 Nuclear PKM2 is a Dimeric Form 153

Figure 3.7 Tyrosine-phosphorylated p68 RNA Helicase Interacts with PKM2

both in vivo and in vitro 155

xvi

Figure 3.8 p68 RNA Helicase Interacts with PKM2 at Its FBP Binding Site 157

Figure 3.9 PKM2, Stat3 and p68 Forms a Protein Complex 159

Figure 4.1 HA-PKM2 Expression Induces Multi Nuclei in SW480 Cells 178

Figure 4.2 Cdc14A Expression is Up-regulated by PKM2 and p68 RNA Helicase 180

Figure 4.3 Nuclear PKM2 Regulates Cdc14A Expression 182

Figure 4.4 Cdc14A Expression is Co-regulated by PKM2 and p68 RNA Helicase 184

Figure 4.5 PKM2 Binds to Cdc14A Promoter Region 186

Figure 4.6 Cdc14A Transcription is Regulated by PKM2 Expression 188

Figure 4.7 Cdc14A Expression in Cancer Cell Lines 190

Figure 7.1.1 P68 Post-translation Modifications Analysis with MALDI-TOF 245

Figure 7.1.2 2-D Gel Analyses of p68-associated Nuclear Proteins 247

Immunoprecipitated with p68 Antibodies

Figure 7.1.3 2-D Gel Analysis of p68-associated Nuclear Proteins Pulled Down 249

with Phophorylated Peptides Derived from p68

1 CHAPTER I GENERAL INTRODUCTION Cancer is a serious human disease and the detail mechanism is not well understoond. Cancer cells uptake more glucose to provide energy and phosphoryl intermediates for macromolecule systhesis, and their oxidative phosphorylation activity is decreased, so-called Warburg effect. Warburg effect has been known for decades and the detail mechanisms are not well defined. In this project, we studied how a glycolytic protein played a role to regulate gene transcription in cells. Moreover, we investigated how phosphorylated proteins affect the glycolytic process by regulating the quaternary structure of a glycolytic enzyme, which provided part explanations for Warburg effect.

1.1 Cancer Metabolism 1.1.1 Tumors and Cancer In general, tumors are the outcome of abnormal growth of normal cells. There are three continuous stages normal cells go through to completely develop into tumor cells. The first stage is hyperplasia, and at this stage the cells grow out of control but still behave like normally physiological cells. At this stage, tumors can be removed by surgery; also at this point they are called benign tumors. The second stage is dysplasia, which is also called the early form of cancerous lesions. At this stage, cells change their behavior and the differentiation and mature processes of cells are delayed. However, cells are not invasive to the basement membrane of the soft tissues. Dysplasic cells can be further graded into different levels even though cells at the highest level of this stage are still considered very low level cancers. The third stage is anaplasia, which is a reversion

2 process of cell differentiation. At this stage, cells lose their functional and structural differentiations at physiological conditions. The nuclei of anaplasic cells become astoundingly hyper-chromatic and large. The nuclear-cytoplasmic ratio increases to 1:1 instead of 1:4~1:6 for normal cells. At this stage, cells gain the ability to invade the blood system as well as neighboring tissues, which are called malignant tumors; these are also defined as cancers. According to their original tissue location, cancers are further divided into five groups: Carcinoma is originally generated from epithelial cells covering the surface of internal organs. Sarcoma is a cancer initially from muscle, bone or connective tissues. Leukemia is generated from white blood cells. Lymphoma is originally from lymphatic cells. Myeloma is from mature B lymphocyte. Cancer cells are characteristically different from normal cells. For example, the disruption of cytoskeletons in cancer cells, such as microfilaments and microtubules, changes their interactions with neighboring cells as well as their appearances. The change in cell to cell adhesion helps cancer cells to lose the restrictions of contact inhibition so that cancer cells can grow unlimitedly even surrounded by other cells. The secretion of enzymes from cancer cells facilitates them in degrading the basement membrane to invade into deep tissues. Therefore, these changes create six functional characteristics for cancer cells with the presence of variously genetic alterations, such as avoidance of apoptosis, constant reaction to growth signals or stimuli, unrestricted replicative capability, angiogenesis capacity, invasion ability and metastasis potential.

3

1.1.2 Cancer Metabolism and Warburg Effect The metabolism in cancer cells is, to a certain extent, different from normal cells (Merida and Avila-Flores 2006). One important characteristic of cancer cell metabolism is the consistent switch of the energy production pathway from oxidative phosphorylation to glycolysis (Kondoh, Lleonart et al. 2007). In most normal mammalian cells, glycolysis flux is tightly regulated, and the glycolysis process is inhibited by the presence of oxygen, which was first discovered by Louis Pasteur. For decades, it has been known that cancer cells utilize more glucose and display a higher glycolysis rate than normal cells. In the 1920s, Otto Warburg found that even in the presence of sufficient oxygen, cancer cells consume glucose for ATP production instead of using the oxidative phosphorylation pathway (Warburg 1956; Warburg 1956). In general, through the glycolysis process, one molecule of glucose generates two molecules of pyruvate and produces two molecules of ATP. In mammals, the product of the glycolysis process, pyruvate, has diverse fates. In an environment with limited oxygen, pyruvate is catalyzed into lactate by lactate dehydrogenase, a process which is called anaerobic glycolysis. In the presence of efficient oxygen, pyruvate is catalyzed into acetyl coenzyme A by the pyruvate dehydrogenase complex. Acetyl coenzyme A is consumed in the citric acid cycle to generate electrons in the mitochondria. The electrons create an electron gradient which generates a pH gradient across the mitochondrial membrane. The force motive produced by the pH gradient generates ATP by ATP synthases. This process is called aerobic oxidative phosphorylation. Overall, one acetyl CoA molecule through citric acid cycle produces two molecules of NADH, one molecule

4 of FADH2, and eight molecules of electrons. Compared to the aerobic oxidative phosphorylation pathway, anaerobic glycolysis only produces a small fraction of the total energy of glucose. One molecule of glucose can be completed degraded into H 2 O and CO 2 in the oxidative phosphorylation process to produce 38 molecules of ATP. Overall, the oxidative phosphorylation process is more efficient in producing ATP than glycolysis. However, cancer cells desire the glycolysis process to produce ATP instead of the oxidative phosphorylation pathway. The reason why cancer cells prefer consuming glucose to produce ATP is not well defined yet. One fact is that the glycolysis process provides a lot of phosphointermediates besides ATP for cells to synthesize macromolecules such as lipids, proteins, and nucleic acids.

1.2 Possible Causes of Metabolic Changes in Cancer Cells 1.2.1 Tumor Microenvironment Affects Tumor Metabolism The metabolism in cancer cells is regulated by many effectors at diverse levels. Firstly, tumor microenvironment is, to a certain extent, different from normal cells, characterized by being surrounded with disorganized microvasculature (Allinen, Beroukhim et al. 2004; Joyce 2005; Anderson, Weaver et al. 2006; Fukumura and Jain 2007; Mohla 2007). Tissues of human bodies need nutrients and oxygen to keep their normal functions, which is provided through surrounding capillary vessels. Even though the growth and proliferation of cancer cells is not totally controlled by the same mechanisms as normal cells, cancer cells still need nutrients as well as oxygen provided by blood vessels. In most conditions, cancer cells proliferate and grow so fast that the

5 surrounding blood vessels can not provide enough oxygen or other nutrients to them. Under these conditions, the solid tumor grows in a hypoxic environment (normally below 3%–5% O 2 ). Without enough oxygen provided to the cancer cells they begin to rely on glycolysis to produce ATP as the major energy providing source. Meanwhile, the hypoxic environment surrounding cancer cells induces continuous expression and activation of hypoxia-inducible transcription factor (HIF-1) to regulate the expression of glycolytic enzymes as well as glucose transporters. Under anoxic conditions, HIF-1 is degraded by proteasomes due to the recognition by the von Hippel-Lindau (VHL) tumor suppressor (Mahon, Hirota et al. 2001). Under hypoxic conditions, HIF-1 is activated and translocates into the nucleus and binds to β-subunit of aryl hydrocarbon receptor nuclear translocator (ARNT) to regulate O 2 -induced gene transcriptions (Wiesener and Maxwell 2003). Nearly all the expression of the glycolysis enzymes, such as hexokinase, phosphofructokinase, aldolase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, enolase, pyruvate kinase, lactate dehydrogenase is regulated by HIF-1 activity (Semenza 2007). The overexpression of these glycolysis enzymes enhances the glycolysis process to provide more energy to cancer cells. On the other hand, under hypoxic conditions, HIF-1 induces VEGF expression from cancer cells which facilitates the growth of blood vessels to provide more oxygen. However, the blood vessels surrounding tumor cells are so disorganized that they can not provide enough oxygen and nutrients as in normal physiological conditions. Under these conditions, the blood vessels surrounding tumor cells still can not alleviate the hypoxic levels of cancer cells. Therefore, aerobic glycolysis is still dominant in these tumor cells.

6

1.2.2 Oncogenes and Tumor Suppressor Genes Regulate Cancer Cell Metabolism Some oncogenes and tumor suppressor genes regulate the glycolysis process in cancer cells, such as c-Myc, AKT, AMPK, Ras and p53. Akt, a Serine/Theronine kinase, was initially discovered as a cellular homolog of a viral oncoprotein; this protein is amplified in diverse cancers, such as ovarian, breast carcinomas, and gastric adenocarcinoma (Vivanco and Sawyers 2002). Akt is the downstream target of phosphatidylinositide-3-kinase (PI-3 kinase) in growth factor induced signal pathways. Under the stimulation of diverse survival signals such as IL-6 and IGF-1, PI-3K is activated by phosphorylation to generate phosphoinositides. Akt is then recruited by phosphoinositides to the cellular membrane to be phosphorylated at Serine 473 and Threonine 308 residues (Kitamura, Kitamura et al. 1999). Akt can be dephophorylated by protein phosphatase 2A (PP2A). The PI3K-Akt pathway is an important signal transduction pathway, playing crucial roles in regulating the cell cycle, apoptosis, cell survival and cell proliferation (Thompson and Thompson 2004). Cellular Akt is also activated by the deletion of phosphatase and tensin homolog on chromosome 10 (PTEN), an antagonist of PI-3, in many cancers, such as brain, breast, and prostate cancers (Li, Yen et al. 1997). In addition, Akt is activated by a number of oncoproteins, such as Bcr-Abl in chronic myelogenous leukemia, Her2 in breast cancer, as well as Ras. Activated Akt has many downstream targets, such as nuclear factor-kappa B (NF- B), GSK-3, and Bad. Activated Akt is pluripotent in contributing to tumorigenisis, such as avoiding apoptosis and promoting cell proliferation. In leukemia cells, the overexpression

7 of Akt promotes its survival capability by inceasing glycolysis levels. Akt regulates glycolysis though many aspects. Firstly, the activated Akt pathway in respiration deficient cancer cells increases glucose consumption by regulating the transcription and translation of glucose transporter1 (GLUT1) to support their survival and growth (Plas, Talapatra et al. 2001; Rathmell, Fox et al. 2003). Second, activated Akt stimulates hexokinase activity to convert glucose to glucose-6-phosphate, a modified form of glucose which can not diffuse freely out of the cell, so as to increase the cellular amount of glucose (Gottlob, Majewski et al. 2001) (Rathmell, Fox et al. 2003). Third, Akt regulates phosphofructokinase (PFK-1) activity, a key enzyme in controlling the glycolysis rate, through phosphorylating and activating PFK-2. PFK-2 catalyze the production of fructose 2,6-bisphosphate, which is an allosteric regulator of PFK-1 to overcome the inhibition effect of high ATP levels on PFK-1 (Deprez, Vertommen et al. 1997). In addition, it is reported that the overexpression of Akt is sufficient to promote transformed cells’ aerobic glycolysis without affecting their oxidative phosphorylation level (Elstrom, 2004 #5019). Furthermore, the inactivation of mitochondrial respiration in human leukemia and lymphoma cell activates PI-3K-Akt pathway by oxidating the tumor suppressor PTEN. The activated PI-3K-Akt pathway benefits cell survival (Pelicano, Xu et al. 2006). The direct effect of Akt on glycolysis may contribute to the Warburg effect (Coloff and Rathmell 2006). c-Myc is a nuclear oncoprotein, which is constantly active in many malignant tumor cells independent of growth factor stimulations (Elliott, Ge et al. 2000). It is reported that c-Myc regulates many glycolysis-related gene transcriptions to control energy production, such as glucose transporters, hexokinase, phosphoglucose isomerase,

8 phosphofructokinase, aldolase, enolase and pyruvate kinase (Gordan, Bertout et al. 2007). Interestingly, c-Myc not only upregulates glycolysis-related genes transcriptions for energy production, but also promotes mitochondrial biosynthesis through targeting the mitochondrial transcription factor A (TFAM) (Li, Wang et al. 2005). The products from mitochondrial biosynthesis provide intermediates for macromolecular synthesis, such as fatty acid, nucleotide, polyamine as well as amino acids (Coller et al., 2000; O’Connell et al., 2003). AMP-activated protein kinase (AMPK), is a master regulator controlling energy metabolism, which was first identified in 1994 as an inhibitor of acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) (Kemp, Stapleton et al. 2003). AMPK activity is stimulated by its upstream effetor, AMPK kinases (AMPKKs), with the phosphorylation at Threonine172 residue. AMPK is also directly activated by AMP. AMPK is reported to regulate glucose consumption by controlling the transcription of GLUT1 and the translocation of GLUT1 to cellular surface (Russell, Bergeron et al. 1999). AMPK activates 6-phosphofructo-2-kinase (PFK-2), the enzyme that synthesizes fructose 2,6-bisphosphate in cardiac muscle cells under ischaemic conditions (Marsin, Bertrand et al. 2000). p53, an important tumor suppressor, plays important roles in regulating apoptosis, cell cycle arrest, and DNA replication. p53 is inactive in more than half of cancers (Hainaut and Hollstein 2000). It is reported that the activation of p53 causes the downregulation of several glycolytic enzymes’ transcription, such as phosphoglycerate mutase (PGM), and GLUT (Schwartzenberg-Bar-Yoseph, Armoni et al. 2004). In the glycolysis process, PGM converts 3-phosphoglycerate to 2-phosphoglycerate to decrease

9 the glycolysis flux. Interestingly, the activation of p53 increases the oxidative phosphorylation rate by upregulation of the synthesis of cytochrome oxidase 2 (SCO2). SOC2 is crucial for regulating cytochrome c oxidase (COX) complex activity to use oxygen in oxidative phosphorylation pathway. Therefore, the loss of p53 activity benefits cell proliferation by increasing the glycolysis rate and decreasing mitochondrial oxidative phosphorylation activity, which is another possible explanation for the Warburg effect (Matoba, Kang et al. 2006). p53 is a nuclecytoplasmic shuttling protein which depends on its leucine-rich nuclear export signal (Stommel, Marchenko et al. 1999). It is reported that p53 also translocates to mitochondria under stimuli to induce apoptosis by directly interacting with a Bcl-2 family protein (Erster, Mihara et al. 2004). Moreover, p53 plays important functions in maintaining mtDNA integrity in response to mtDNA damage by interacting with mtDNA and DNA polymerase γ to enhance poly γ transcriptional activity (Achanta, Sasaki et al. 2005).

1.2.3 Mitochondrial Malfunction in Cancer Cells Mitochondrion is an important cellular organelle which takes part in regulating cell metabolism, producing reactive oxygen species (ROS) as well as inducing apoptosis (Lu, Sharma et al. 2009). Mitochondria have their own DNA as well as DNA transcription, replication and repair system to encode 13 polypeptides as the components of oxidative phosphorylation chain (Anderson, Bankier et al. 1981). In cancer cells the energy producer function of mitochondria is decreased for many reasons. First, in cancer cells, the transcription rate of the mitochondrial genome is reduced to a certain level compared to normal cells (Gogvadze, Orrenius et al. 2008; Bellance, Lestienne et al. 2009). Second,

10 mtDNA lacks histone protection from damages. Third, mitochondrial DNA is circular, supercoiled, and is easily damaged due to diverse exogenous or endogenous effects, such as radiation and ROS. Fourth, there are few DNA repair mechanisms in mitochondria compared to the nuclear genome (Achanta, Sasaki et al. 2005). All these properties make mitochondrial DNA to be easily affected by mutations, which disrupts the electron transport chain to decrease mitochondrial respiration rate. In addition, the malfunction of the electron transport chain in cancer cells produces superoxide, which forms ROS with other radicals. The accumulation of ROS damage mtDNA and nuclear DNA causes genetic instability as well as cancer progression (Pelicano, Carney et al. 2004). Usually, there are two characteristics of mtichondrial DNA mutations in cancer cells. First, the mutations are base transitions from T to C or G to A. Second, the mutations in the mitochondrial genome frequently occur in the 1.1 kb displacement-loop region (Ding, Ji et al. 2009). The malfunction of mitochondrial genome decreases oxidative phosphorylation efficiency for ATP production. On the other hand ATP produced in the oxidative phosphorylation process is an inhibitor for phosphofructokinase 1 (PFK1) in glycolysis. PFK1 is the major pace-keeping enzyme in glycolysis, and activated PFK1 increases glycolysis flux, which makes a transition for energy production from oxidative phosphorylation to glycolysis in cancer cells. Moreover, studies show that the disruption of mitochondrial respiration function in cancer cells induces tumorigenesis. For example, the interruption of mitochondrial respiration with genetic, chemical and microenvironmental methods, activates the PI3K-Akt pathway (Robey and Hay 2009). It is well known that the PI3K-Akt pathway in cancer cells plays important functional roles

11 in cell survival, proliferation and migrations (Robey and Hay 2009). In mitochondria- deficient human leukemia and lymphoma cells, glycogen synthase kinase-3 (GSK-3), the substrate of Akt, is activated by Akt phoshphorylation. Therefore, the malfunctions of mitochondrial respiration in cancer cells may present part of the full explanation for the Warburg effect.

1.2.4 Metabolic Malfunction is a Potential Cancer Therapy Method. Even though the mechanisms of the Warburg effect are not fully understood, thus far, the theory of the Warburg effect has been applied clinically in cancer diagnosis and therapy. For example, the Warburg effect has been used in cancer imaging for tumor detection by a glucose analogue, fluorodeoxyglucose, Positron Emission Tomography (FdG-PET) (Weber, Schwaiger et al. 2000). The fact that cancer cells require more glucose for energy production leads to FdG-PET, a powerful method in cancer diagnosis and prognosis (Kelloff, Hoffman et al. 2005). In addition, the increase of protein expression in glycolysis in cancer cells, such as HK2, and the decrease of the expression of components in the oxidative phosphorylation pathway, such as β-F1-ATPase, are also applied as biomarkers for cancer diagnosis (Cuezva, Ortega et al. 2009). On the other hand, Akt/mTOR/HIF-1, a major regulatory signal pathway for the Warburg effect, has been targeted as a cancer therapy method to switch the energy production sources, that is, to decrease glycolysis efficiency and increase oxaditive phosphorylation competence (Jiang and Liu 2008). The activation of HIF-1 induced by Akt under hypoxia environment promotes cancer cell survival. By these methods, the survival advantages of cancer cells in hypoxic environments will be removed so that the

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Abstract: Cancer cells take more glucose to provide energy and phosphoryl intermediates for cancer progression. Meanwhile, energy-provider function of mitochondria in cancer cells is disrupted. This phenomenon is so-called Warburg effect, which is discovered over eighty years ago. The detail mechanisms for Warburg effect are not well defined. How glycolytic enzymes contribute to cancer progression is not well known. PKM2 is a glycolytic enzyme dominantly localized in the cytosol, catalyzing the production of ATP from PEP. In this study, we discovered that there were more nuclear PKM2 expressed in highly proliferative cancer cells. The nuclear PKM2 levels are correlated with cell proliferation rates. According to our microarray analyses, MEK5 gene was upregulated in PKM2 overexpression cells. Our studies showed that PKM2 regulated MEK5 gene transcription to promote cell proliferation. Moreover, nuclear PKM2 phosphorylated Stat3 at Y705 site using PEP as a phosphoryl group donor to regulate MEK5 gene transcription. Our study also showed that double phosphorylated p68 RNA helicase at Y593/595 interacted with PKM2 at its FBP binding site. Under the stimulation of growth factors, p68 interacted with PKM2 to promote the conversion from tetrameric to dimeric form so as to regulate its protein kinase activity. Overexpression PKM2 in less aggressive cancer cells induced the formation of multinuclei by regulating Cdc14A gene transcription. Overall, this study presents a step forward in understanding the Warburg effect. Index words. PKM2, P68 RNA helicase, MEK5, Stat3, Cdc14A, Tyrosine phosphorylation, Gene transcription, Cell cycle, Glycolysis.