Luận án: Vai trò của PPARγ trong Chuyển hóa Ung thư Phổi và Glioblastoma

Peroxisome Proliferator-Activated Receptor Gamma-Mediated Cancer Metabolism in Lung and Glioblastoma Tuyen Ngoc Minh Hua Department of Global Medical Science The Graduate School Yonsei University Peroxisome Proliferator-Activated Receptor Gamma-Mediated Cancer Metabolism in Lung and Glioblastoma Directed by Professor Yangsik Jeong A Doctoral Dissertation Submitted to the Department of Global Medical Science and the Graduate School of Yonsei University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Science Tuyen Ngoc Minh Hua August 2019 ACKNOWLEDGEMENT Ph. is a valuable journey that I will never forget in my life. Being a graduate student in a foreign country has taught me more than what I can imagine. There are many “first times” to memorize during my course.

The first time I was far away from my parents for a long time, I know how important they mean to me. The first time I struggled to make a decision, I learn about responsibility for what I have chosen. The first time I have a chance to look back myself seriously before continuing to go on my own way. I believe that everything happens in life for a reason.

I am thankful for all to grow me up by your encouragements, belief and love despite challenges and difficulties. I would like to express all of my appreciation and gratitude to my advisor, Professor Yangsik Jeong for his patience, dedication and motivation. He has taught me how to become strong and brave to face and overcome hardship. Without his supports and guidance, this project will never be achieved.

I am really thankful to the committee members for their valuable comments and validation of the research project: Professor Kyu-Sang Park, Professor Won Gil Cho, Professor Seung-Kuy Cha and Professor Jun Namkung. This thesis would not be finished without their continuous supports. I am sincerely grateful to all the members in my Department of Biochemistry, Professor Jong-Whan Choi, Professor Hyun-Won Kim, Professor Jun Namkung, Jiyeon Om, Jihye Kim and Jiyoung Choi for their helps and supports during my Ph. I would also like to appreciate Doctor Ji-woong Oh, Doctor So-hyun Kim and Doctor Myung-Jin Park for their precious collaboration in my research.

I would like to send my all thanks to my Vietnamese seniors, Doctor Nguyen Thi Tuyet, Doctor Doan Van Khanh and Doctor Phan Nguyen Hoa Ai for the conscientious instructions and supports from the very beginning days that I was in Korea, not only in research but also in life. I really appreciate my lab members, Min-Kyu Kim for his help in both cancer research and Korean life, Vo Thi Anh Vu for being my constant companion and for thesis revision, Nguyen Thai Anh Thu and Jayson M. Antonio for staying with me in this journey. To Vu, the time here becomes fulfilling, bright and meaningful with your presence as the special gift for my life.

I would like to thank all of my Vietnamese friends for our happy memories that we had together, Doctor Luong, My Khanh, Da Dat, Nhung Nang, Phan Anh, Bao, Doctor Ha, Hanh and my fellow students from international student group, department of Physiology, Pharmacology and MRC center for their warmness and kindness. Finally, all of my thanks, love and gratitude for my parents, the greatest heroes in my mind. Thank you for always thinking for me and absolutely loving me. Special thanks to my maternal grandfather, uncles, aunts and cousins for your care not only for me but also for my parents when I am not in Vietnam.

For my paternal grandfather, paternal grandmother and maternal grandmother, I am sorry not being with you in your final minutes but you are always in my heart. April 17th, 2019 Tuyen Ngoc Minh Hua TABLES OF CONTENTS LIST OF FIGURES. vi LIST OF TABLES. ix LIST OF ABBREVIATIONS.

PPAR suppresses ALDH1A3 to inhibit lung cancer progression. Peroxisome proliferator-activated receptor gamma (PPAR). Structure of PPAR. Endogenous ligands of PPAR.

Thiazolidinediones (TZDs) as synthetic ligands of PPAR. General functions of PPAR. Functions of PPAR in lung cancer. Aldehyde dehydrogenases 1 family member A3 (ALDH1A3).

MATERIALS AND METHODS. Cell culture and reagents. Microarray data analysis. PPAR binding site screen.

Chromatin immunoprecipitation (ChIP) assay. Relative RT-PCR analysis. Expressional correlation between ALDH1A3 to PPARG. ALDH1A3 is an inverse target of PPAR.

ALDH suppression decreases lung cancer cell proliferation. Inhibition of ALDH1A3 by pioglitazone induces lipid peroxidation. Lung cancer lipid metabolism regulation by PPAR – FABP4 is mediated by oncogene Src. Reprogramming lipid metabolism in cancer.

Lipid biosynthesis in cancer cells. Fatty acid catabolism in cancer cells. Lipid droplets as protective factor for cancer cells. Lipid rafts in tumor.

Non-receptor tyrosine kinase Src. Fatty acid-binding protein 4 (FABP4). MATERIALS AND METHODS. Cell culture and reagents.

Immunoblot and immunoprecipitation assays. Relative RT-PCR analysis. Oil-red O (ORO) staining. Cell growth assay.

Intracellular ROS measurement. Oxygen consumption rate measurement. Gene expression analysis. Regulation of Src on lipid content in lung cancer cells.

Inhibition of Src increased PPAR-induced FABP4. Src suppresses PPAR activity in a PPAR-Tyr 78 and Src kinase activity independent manner. Src inhibition reduces lipid content in an FABP4 dependent manner in in vitro and in vivo model. FABP4-dependent accumulation of intracellular ROS upon Src suppression.

Lipolytic genes serve as prognostic factors in lung and renal cancer. PPAR is a novel target for mesenchymal glioblastoma treatment. Cellular heterogeneity of glioblastoma. Brain tumor stem cells.

Phenotypic diversity of glioblastoma stem cell subtypes. Current treatment strategies in GBM. Treatment for newly diagnosed GBM .2 Treatment for recurred GBM. PPAR agonist as potential therapy in GBM.

Brain tumor growth inhibition. Induction of glioma differentiation. Local invasiveness reduction. PPAR agonist in clinical study.

MATERIALS AND METHODS. Cell culture and reagents. RNA-seq analysis. Relative RT-PCR analysis.

Gene expression analysis. PPAR knockdown using siRNA. Cell growth assay. In vitro limiting dilution assay and sphere forming assay.

Patient sample collection and preparation. PPAR is exclusively expressed in Mes GSCs. Evaluation of endogenous PPAR function in Mes GSCs. Gain of function studies for PPAR reveals its tumor suppressor role in PN GSCs.

PPAR is therapeutic target for Mes GBM in vitro. High expression of PPAR in Mes type of GBM patients. 127 ABSTRACT IN KOREAN. 155 v LIST OF FIGURES Figure 1.

Positive correlation between ALDH1A3 and PPARG expression in various lung cancer cell lines. ALDH1A3 is regulated by PPAR. ALDH1A3 is suppressed by PPAR. PPAR activator shows the same growth inhibitory effect with ALDH inhibition.

PPAR activation increases lipid peroxidation and reduces cell growth. Proposed model of the molecular interaction between PPAR and ALDH1A3. Involvement of Src in lipid metabolism. Src negatively correlates with FABP4 and LPL.

FABP4 expression in response to Src inhibitor treatment. FABP4 mRNA and protein expression upon Src knockdown in Calu6 cells transfected with siControl (siCtr) or siSrc for 48 hours. Src suppressed FABP4 expression. FABP4 is PPAR’s target gene.

FABP4 expression upon Src inhibition in HBEC-C1-PPAR (A) or H1299 (B) cells. Suppression of PPAR transcriptional activity evaluated by luciferase assay with PPRE3x-luc construct. Src specific-suppression of PPAR. PPAR activity in various conditions including PPARY78F construct with wtSrc-GFP (A) or wt-PPAR with Src mutation (B and C).

PPAR activity suppressed by Src is not dependent on Src kinase activity and Stat3. Src interacts with PPAR. Lipid content (left) and quantification (right) in HBEC-C1-PPAR (upper) and Calu6 (lower) cells. Lipid content upon Src knockdown in Calu6.

Lipid content upon Src knockdown with or without FABP4 knockdown in Calu6. Src inhibition decreases cancer growth in vitro and in vivo. Relative tumor volume of individual mice. Lipid content and FABP4 expression in xenograft tumor upon SU6656 treatment.

ROS measurement in response to Src and/or FABP4 regulation. AMPK phosphorylation and 4HNE adducts upon Src and/or FABP4 inhibitor treatment. Evaluation of oxidative phosphorylation upon Src and/or FABP4 inhibitor treatment. Cell viability in response to Src and/or FABP4 regulation.

Expression of Src and lipid regulators in patient tissues. FABP4 and LPL link to better prognosis in patients with lung and renal cancer. FABP4 and LPL link to better prognosis in patients with lung cancer. Hypothetical model of Src regulation on lipid content in lung cancer.

Nuclear receptor expression in Mesenchymal (Mes) and Proneural (PN) glioma stem cells (GSCs). Expression of PN and Mes markers in GSCs. PPAR expression in PN and Mes GSCs. PPAR upregulation in Mes GBM patients.

Cell viability under pioglitazone treatment in GSCs. Effect of pioglitazone on self-renewal ability of PN and Mes GSCs. Effect of pioglitazone treatment on Stat3 signaling in PN and Mes GSCs. Effect of PPAR knockdown on cell viability in Mes GSCs.

Sphere formation in PN GSCs with PPAR overexpression and activation. Expression of mRNA in PN GSCs with PPAR overexpression and activation. Expression of mRNA in PN GSCs with PPAR overexpression and activation. Forced PPAR expression attenuates Proneural Mesenchymal Transition (PMT).

Tumor growth suppression upon pioglitazone treatment. High PPAR expression links to poor outcome of GBM patients analyzed by Kaplan-Meier plot. Expression of PPAR, SOX2 and CD44 in microdissected GBM tissues. Expression of PPAR, SOX2 and CD44 in primary and recurred GBM tumors.

PPAR, SOX2 and CD44 expression in patients tissues. Hypothetical model of PPAR function as therapeutic target in GBM. 122 viii LIST OF TABLES Table 1. Primer sequences for ChIP assay.

Primer sequences for RT-PCR. List of 78 lung cancer cell lines used to analyze Pearson correlation. Pearson correlation of PPARG to individual ALDHs. Primer sequences for site-directed mutagenesis.

Target sequences of siRNA. Primer sequences for RT-PCR. Analysis of protein expression in lung cancer patients. Primer sequences for RT-PCR.

siRNA sequences of PPAR. Stem cell frequency of Proneural (PN) or Mesenchymal (Mes) cells treated with pioglitazone .