Studies on adipose stem cells and ovarian cancer stem cells

다국어 초록 (Multilingual Abstract)

The problems in ovarian cancer therapy are that ovarian cancer is diagnosed at an advanced stage and resistant to chemotherapy, leading to poor prognosis. Adipose stem cells (ASCs) and cancer stem cells (CSCs) can be driving forces acting on ovarian cancer progression and chemoresistance, respectively. The present study focused on the interaction between ASCs and ovarian cancer, and the role of 2) CSCs in ovarian cancer. First, ASCs regulate obesity, defined as abnormal or excessive fat accumulation, through the proliferation and adipogenic differentiation, and contribute tumor microenvironment supporting cancer cells. Although several epidemiologic studies have suggested evidence for the relationship between obesity and ovarian cancer, experimental evidence is still lack to verify it. Here, we suggested that ASCs affect ovarian cancer progression via paracrine factors. Among adipokines containing in the conditioned medium of ASCs, interleukin-6 (IL-6) strongly expressed and facilitated the migration and proliferation of ovarian cancer cells (SKOV3 and patient-derived cancer cells). IL-6 derived from ASCs activated the JAK2/STAT3 signaling pathway of ovarian cancer cells, leading to enhanced cancer progression. Taken together, ASCs as a part of tumor microenvironment facilitate the migration and proliferation of ovarian cancer cells via secretion of the paracrine factor. This result provides experimental evidence for the relationship between obesity and ovarian cancer. Second, existence of CSCs with self-renewal and clonal initiating potential confers chemoresistance to ovarian cancer through the multiple way, such as drug efflux, detoxification (drug metabolism), and dormancy. Sphere formation induced enrichment of CSCs with high ALDH activity. CSCs exhibited expression of ABC transporter proteins (MDR1/ABCB1 and ABCG2), and were resistant to cisplatin compared to parental cells (attached-cultured cells). Oxidative stress caused by sphere formation induced PGC-1α expression (a key regulator of mitochondrial biogenesis and metabolism), resulting in the enhanced chemoresistance of sphere forming cells. When oxidative stress was exogenously exerted to parental cells, PGC-1α expression was observed. Overexpression of PGC-1α in parental cells led to the same response with sphere forming cells. Silencing of PGC-1α in sphere forming cells reduced the resistance to cisplatin. Collectively, PGC-1α induced by oxidative stress mediates chemoresistance in ovarian cancer. We first reported a new function of PGC-1α for the intermediate mechanism in chemoresistance. Therefore, targeting the microenvironmental effects of ASCs and chemoresistant properties of CSCs could elicit a better response to therapy and prognosis in ovarian cancer.

목차 (Table of Contents)

• ABSTRACT········································································i
• CONTENTS······································································iii
• LIST OF FIGURES·························································· vii
• LIST OF ABBREVIATIONS··············································ix
• GENERAL INTRODUCTION············································1
• Chapter I. Adipose stem cells and cancer······································3
• Chapter II. Cancer stem cells and chemoresistance·························7
• CHAPTER I·····································································12
• Adipose stem cells from visceral and subcutaneous fat depots enhance growth and migration of ovarian cancer cells via activation of IL-6/JAK2/STAT3 pathway
• Abstract············································································13
• Introduction·······································································14
• Materials and Methods························································· 15
• - Isolation of CD45-/CD31- ASCs··········································16
• - Culture of ASCs and ovarian cancer cells·······························17
• - Isolation and culture of ascites cells from ovarian cancer patients·······································································17
• - Immunophenotype of ASCs···············································18
• - In vitro differentiation of ASCs into adipogenic, chondrogenic, and osteogenic lineage··························································18
• - Proliferation assay··························································19
• - Wound healing assay·······················································20
• - Boyden chamber assay ····················································20
• - Depletion of secreted IL-6 ················································21
• - Western blotting·····························································21
• - Statistical analysis·························································· 22
• Results············································································· 22
• - ASCs isolated from subcutaneous and visceral fat depots share similar features to mesenchymal stem cells ···························· 22
• - ASC-CM enhance the migratory ability of ovarian cancer cells with no difference between S- and V-ASC-CM······························ 23
• - Of the several factors including IL-6, TNF-α, adiponectin, and leptin, only IL-6 enhances the migration of ovarian cancer ·················· 23
• - JAK2/STAT3 signaling is activated by IL-6 secreted from ASCs in ovarian cancer cells·························································24
• - IL-6/JAK2/STAT3 signaling pathway activated by ASCs also enhances proliferation and migration of ascites cells isolated from ovarian cancer patients·····················································25
• Discussion··········································································26
• CHAPTER II····································································44
• PGC-1α under oxidative stress mediates chemoresistance of sphere forming cells in ovarian cancer
• Abstract············································································45 Introduction·······································································47
• Materials and Methods························································· 49
• - Cell culture of ovarian cancer cells·······································49
• - Sphere formation of ovarian cancer cells································49
• - Measurement of ALDH activity·········································· 49
• - Measurement of intracellular ROS levels in ovarian cancer cells····50
• - Total RNA isolation and quantitative real-time PCR (qRT-PCR)···· 50
• - Western blotting·····························································51
• - Relative cell viability using MTT assay································· 52
• - Measurement of apoptosis·················································52
• - Immunocytochemistry for the detection of PGC-1α in tumor spheres········································································52
• - Statistical analysis·························································· 54
• Results··············································································55
• - Tumor sphere formation increases cancer stem-like cell population and exhibits enhanced drug-resistance in ovarian cancer cells·······55
• - Tumor sphere formation stimulates ROS generation, related to stem-like phenotype of ovarian cancer cells···································55
• - Intracellular ROS generated in tumor spheres lead to PGC-1α expression····································································56
• - PGC-1α mediates drug-resistance in tumor sphere cells·············· 57
• Discussion ·········································································58
• GENERAL CONCLUSION···············································75
• REFERENCES·································································78
• 국문 초록··········································································102