EFFECTS of ACSVL3 KNOCKOUT on LIPID and GLUCOSE METABOLISM in MALIGNANT GLIOMA CELLS by Elizabeth Anne Kolar a Dissertation Subm
Total Page：16
File Type：pdf, Size：1020Kb
EFFECTS OF ACSVL3 KNOCKOUT ON LIPID AND GLUCOSE METABOLISM IN MALIGNANT GLIOMA CELLS By Elizabeth Anne Kolar A dissertation submitted to The Johns Hopkins University in conformity with the requirements of the degree of Doctor of Philosophy Baltimore, Maryland March 2016 ABSTRACT Gliomas are the largest category of primary central nervous system tumors. Glioblastoma multiforme (GBM) is a World Health Organization Grade IV glioma that comprises 70% of all gliomas. Prognosis is very poor once diagnosed, and current treatments cannot prolong survival after relapse. Very long-chain acyl-CoA synthetase 3 (ACSVL3) is overexpressed in malignant glioma, and depleting ACSVL3 in GBM cells (e.g. U87MG) diminishes their tumorigenic properties and affects signaling through receptor tyrosine kinases. An ACSVL3-deficient knockout (KO) U87MG cell line was generated to study how ACSVL3 contributes to the malignant properties of glioma cells. Acyl-CoA synthetase enzyme activity was measured with long- and very long-chain fatty acids: palmitic acid (C16:0), stearic acid (C18:0), behenic acid (C22:0), and lignoceric acid (C24:0). There were significant decreases in the activation of stearic and behenic acids, while the activation of palmitic acid and lignoceric acid did not change. Ceramide synthesis assays and liquid chromatography/tandem mass spectrometry (LC/MS-MS) analysis revealed a decrease in C18:0 and C22:0 ceramides, reinforcing the enzyme activity assay results. LC/MS-MS analysis also revealed a decrease in sphingosine 1- phosphate, an important signaling molecule that affects growth and proliferation. Proteomic analysis showed lower protein levels for enzymes involved in ceramide synthesis in the ACSVL3 KO line. Fluorescent microscopy and thin layer chromatography analyses show that ACSVL3 deficiency affects lipid rafts and ganglioside synthesis. Proteomic analysis also predicted changes in glycolysis and the tricarboxylic acid cycle (TCA) when ACSVL3 is depleted in U87MG cells. Glycolytic ii enzymes were higher while TCA enzymes were lower in ACSVL3 KO cells. Immunofluoresence using an antibody that detects Tom20, a mitochondrial outer membrane marker, revealed differences in mitochondrial morphology in the ACSVL3 KO cells when compared to the U87MG cells. From these studies, we conclude that ACSVL3 is important for the synthesis of structural and signaling sphingolipids that contribute to the growth and proliferation of the GBM cells, and that this enzyme likely contributes to mitochondrial-involved carbohydrate metabolism. Thesis Advisor: Paul A. Watkins, MD, PhD Thesis Reader: Dan Raben, PhD iii ACKNOWLEDGMENTS There were a lot of people who helped me accomplish this work, whether it was helping with experiments in the lab or encouraging me from home, and I would like to acknowledge those who have made this possible. I came to Hopkins with very specific ideas of what type of research I wanted to do, but as with many things in life, I was led in a very different direction. I never saw myself studying lipid biology, but I found my way to Paul’s lab, and I am very glad that I did. Paul, more than anyone else, knows of the struggles we have had in the lab. Through his encouragement and expertise, I learned so much about science and about myself. I will always be grateful to Paul for all the opportunities and all that he has done. Chapter 1 of this thesis would not have been possible without the collaborative spirit of our lab, and I will always be appreciative of the former and current lab members of the Watkins lab – Zhengtong Pei, Cicely Exeter, Xiaohai Shi, Haiyan Yang, Yanqiu Liu, Xiaoli Ye, and Emily Clay. We got through the struggle with dead/dying/reverting cells together, solved the problem, and characterized the knockout cells as a lab. Above all, I don’t know if I would have made it through graduate school without everyone’s discussions, opinions, hard work, and above all, the laughter and fun they provided every day. Thank you also to my thesis committee – Dan Raben, Will Wong, and Greg Riggins. Their ideas and knowledge helped shape this thesis into what it is. I will always cherish the amazing friends I found here at Hopkins. We came from all over the country – and sometimes from all over the world! – and graduate school iv brought us together. I want to especially acknowledge Marcus Seldin, Michael Multhaup, and Steven Wang for their support and friendship through the years. We have been through a lot, whether it was changing dissertation topics or trying to survive the city with as little damage as possible, we did it together. I want to thank all of my aunts, uncles, and cousins for their support and love. I love you, and thank you for sticking with me through it all. You have all been my rock through this journey. This is for my grandparents, Nicholas and Mary Kolar and Andrew and Patricia Szoke, who mean everything to me. They were always quick with a prayer or novena, and I knew that I always, always had their support. I dedicate this to them, especially to my grandmother, Patricia, who will not be here to celebrate. I know she is always watching over me. I hope I have made her proud. A huge thank you goes to Basil Hussain, without whom I don’t know if this would have been possible. We made it through graduate school together, and there is finally the light at the end of the tunnel. You were a pillar of support through the good and the bad, and I am excited to start our next journey. I love you. Finally, I want to thank my parents and my brother Stephen, who always believed in me. They supported every crazy dream I ever had. Because of them, I realized I could achieve whatever I wanted, especially this. This degree was probably the hardest thing I ever had to do, and even if they didn’t always understand what I was doing and why I was so upset, they were always ones I could lean on. Thank you for everything. v TABLE OF CONTENTS Abstract ii Acknowledgments iv Table of Contents vi List of Figures vii List of Tables x Introduction 1 Chapter 1 Introduction 24 Materials and Methods 26 Results 36 Discussion 52 Chapter 2 Introduction 57 Methods and Materials 61 Results 69 Discussion 92 Chapter 3 Introduction 100 Methods and Materials 104 Results 108 Discussion 130 References 133 Curriculum Vitae 144 vi LIST OF FIGURES Figure 1. Lipid rafts are membrane microdomains enriched with cholesterol, sphingolipids, and proteins………………………………………………………..5 Figure 2. A simplified receptor tyrosine kinase signaling cascade………………………9 Figure 3. The Warburg Effect in proliferating tissues increases lactate through aerobic glycolysis………………………………………………………………………...12 Figure 4. Fatty acid activation reaction………………………………………………....16 Figure 5. Once activated, fatty acyl-CoA molecules can be used by cells in different ways……………………………………………………………………………...17 Figure 6. ACS enzymes can be grouped by substrate specificity in a phylogenetic study……………………………………………………………………………..18 Figure 7. Immunohistochemistry shows that ACSVL3 is overexpressed in glioma……21 Figure 8. ZFNs create a genomic ACSVL3 KO by deleting a 210 bp region…………..39 Figure 9. Proteomic analysis and qPCR show a decrease in ACSVL3 relative to the U87MG cell line………………………………………………………………....40 Figure 10. ACSVL3 KO cells have an altered morphology and grow significantly slower than the U87MG cells……………………………………………………42 Figure 11. ACSVL3 KO cells grow smaller subcutaneous tumors in nude mice………44 Figure 12. Total activation of long- and very long-chain fatty acids is lower in ACSVL3 KO cells……………………………………………………………….49 Figure 13. The majority of ACSVL3 KO cells are in S-phase………………………….51 vii Figure 14. Total cholesterol is not different between U87MG cells and ACSVL3 KO cells………………………………………………………………71 Figure 15. Lipid synthesis is not affected by depletion of ACSVL3 in U87MG cells…73 Figure 16. Ceramide synthesis decreases in ACSVL3 KO cells when the substrate is C18:0……………………………………………………………….76 Figure 17. Ceramdies with acyl chains C18:0-C22:0 are decreased in ACSVL3 KO cells…………………………………………………………………………77 Figure 18. Sphingosine and S1P levels decrease when cells are depleted of ACSVL3………………………………………………………………………..79 Figure 19. Fluorescent microscopy reveals an increase in lipid raft staining in ACSVL3 KO cells………………………………………………………………85 Figure 20. Total gangliosides decrease in ACSVL3 KO cells, but GM1 ganglioside increases compared to U87MG cells……………………………………………87 Figure 21. ACSVL3 KO cells do not increase lipid synthesis when incubated with EGF………………………………………………………………………..90 Figure 22. Decreasing activation of fatty acids destined for sphingolipid synthesis leads to aberrant signaling from receptor tyrosine kinases……………………..99 Figure 23. Glucose uptake in ACSVL3 KO cells relative to U87MG cells…………..110 Figure 24. Glycolytic pathway metabolites do not significantly change despite a change in enzymes……………………………………………………………114 Figure 25. Pyruvate levels do not change with an ACSVL3 KO……………………...115 Figure 26. LDH enzyme levels increase in an ACSVL3 KO, but cellular lactate levels are unchanged……………………………………………………………116 viii Figure 27. Oxidation of glucose is significantly downregulated in the ACSVL3 KO……………………………………………………………………118 Figure 28. TCA Cycle intermediates are not changed overall in the ACSVL3 KO cells……………………………………………………………………………..122 Figure 29. Mitochondrial morphology is different between the U87MG cell line and the ACSVL3 KO cell line………………………………………………………128 ix LIST OF TABLES Table 1. The relative abundance of the long- and very long-chain acyl-CoA synthetases in U87MG cells