The role of ascites tumor-microenvironment in ovarian cancer invasion and chemoresistance

학위논문 (박사)-- 서울대학교 대학원 : 종양생물학전공, 2017. 2. 송용상.Ovarian cancer is the most lethal gynecologic malignancy, because of asymptomatic nature of this disease, most patients are diagnosed in late stage with peritoneal dissemination and distant metastasis. The importance of tumor microenvironment and cancer progression are increasingly recognized and the abnormal accumulation of fluid in the peritoneal cavity, called ascites is found in almost all recurrent ovarian cancer patients. Indeed, the presence of ascites correlates with peritoneal tumor spread and decreased 5-year survival in ovarian cancer. In current studies, we provide the malignant role of ascites in ovarian cancer progression. The cytokine profiles of ovarian cancer patient derived ascites demonstrated the presence of pro-inflammatory cytokines. Of those, a significantly elevated levels of interleukin 6 (IL-6), increased invasion of ovarian cancer cells. Neutralization of IL-6 in ascites reduced the stimulatory effects of ascites on ovarian cancer cell invasion. Ascites increased invasion through JAK2 and STAT3 signaling pathway, confirmed by use of selective inhibitors. Moreover, the expression of IL-6 receptor (IL-6R) on cell membrane of ovarian cancer cells correlated with ascites-induced invasion. Cholesterol is elevated in ascites and treatment of cholesterol reduced response to cisplatin and increased membrane expression of ATP-binding cassette transporters (ABC transporters), via liver x receptor / (LXR/ in ovarian cancer cells. Similarly, ascites treatment reduced response to cisplatin and increased membrane expression of ABC transporters, with increased LXR/ expression. Excess cellular cholesterol is toxic, a feed-forward regulatory system such as the liver x receptor (LXR) family are activated in response to free cholesterol accumulation. Depletion of free cholesterol reduced ascites mediated increased ABC transporter expression and increased response to cisplatin. Our findings highlight the important role of ascites tumor microenvironment in ovarian cancer invasion and chemoresistance. Hence, better understanding of individual components of ascites in ovarian cancer progression will provide novel therapeutic targets as well as prognostic markers.Chapter 1. Introduction 1 1.1 Ovarian cancer 2 1.2 Tumor microenvironment 2 1.3 Ascites as a tumor microenvironment in ovarian cancer 3 1.4 Components of ascites 3 1.5 Ascites tumor-microenvironment contributing to cancer progression and chemoresistance 6 1.6 References 19 Chapter 2. Malignant ascites enhances migratory and invasive properties of ovarian cancer cells with membrane bound IL-6R in vitro 15 Abstract: 16 2.1 Introduction 17 2.2 Materials and Methods 19 2.2.1 Cell culture, clinical samples and reagents 19 2.2.2 Wound healing assay 20 2.2.3 Invasion assay 20 2.2.4 Western Blotting 21 2.2.5 Reagents and Antibodies 21 2.2.6 Ascites analysis using Proteome Profiler cytokine array 21 2.2.7 Determination of IL-6 concentration by ELISA 22 2.2.8 Depletion of soluble interleukin-6 22 2.2.9 RT-PCR 22 2.2.10 Small interfering RNA transfection 23 2.2.11 Immunofluorescence Microscopy 23 2.2.12 Statistical Analysis 24 2.3 Results 25 2.3.1 Ascites promotes migration and invasion of EOC cells 25 2.3.2 High levels of pro-inflammatory cytokines in malignant ascites from patients with ovarian cancer 30 2.3.3 IL-6 in ascites increases migration and invasion via JAK2-STAT3 signaling 35 2.3.4 Ascites increase invasion only in ovarian cancer cells with IL-6R expression on cell membrane 45 2.4. Discussion 54 2.5. References 56 Chapter 3. Cholesterol in malignant ascites enhances chemoresistance via LXR/ in ovarian cancer cells 61 Abstract: 62 3.1 Introduction 63 3.2 Materials and Methods 65 3.2.1 Cell culture, clinical samples and reagents 65 3.2.2 Cell viability assay 65 3.2.3 Cell death analysis 66 3.2.4 Western Blotting 66 3.2.5 Reagents and Antibodies 66 3.2.6 Nuclear and cytoplasmic protein extraction 67 3.2.7 Ascites cholesterol quantitation 67 3.2.8 Statistical Analysis 67 3.3 Results 69 3.3.1 Response of ovarian cancer cell lines to cisplatin is associated with ABC transporter protein expression 69 3.3.2 Cholesterol pre-treatment increase chemoresistance of ovarian cancer cells 72 3.3.3 Cholesterol increase ABC transporter protein expression 75 3.3.4 Cholesterol increase ABC transporters through sterol sensor 78 3.3.5 Ascites cholesterol increase chemoresistance of ovarian cancer cells 81 3.4 Discussion 85 3.5 References 87 Chapter 4. Conclusion 91 4.1 Conclusion 92 4.2 Therapeutic implication of targeting tumor microenvironment 93 4.2.1 Utility of ascites as a diagnostic factor. 93 4.2.2 Personalized therapy 95 4.3 References 96 국문 초록 98Docto