Invasion And Metastasis Of Ovarian Cancer Cells Induced By Lipid Metabolism Controlled By CGI-58 Gene And Its Mechanism Research

Background The metabolic reprogramming is one of the most important characteristics of malignant tumor cells’ features. The lipid metabolic reprogramming plays an important role in growth, proliferation, invasion and metastasis. Lipid not only serves as a store of cell energy, but also an important component of cell organelles. The lipid metabolism process involves a variety of enzymes. The changes of enzyme activity will not only affect tumor lipid metabolism, but also affect the biological characteristics of tumor cells. Among those lipid metabolism-related enzymes, the coenzyme CGI-58, encoded by ABHD5 gene, works as a co-activator of adipose triglyceride lipase(ATGL), assisting hydrolysis of triglycerides into diglycerides and free fatty acids.CGI-58 gene knockout will not only alter the metabolism of tumor cells, especially cell invasion based on our previous research results. Our previous studies demonstrated that the expression level of CGI-58 in tumor cells was correlated with tumor malignance level and tumor cell invasion and metastasis. Transfection of lentiviral sh RNA targeting gene CGI-58 in tumor cells led to changes of capacity of tumor cell invasion and different tumor cell lines showed different levels of changes. Among all tumor cell lines we tested, Colon cancer HCT-116 and ovarian cancer cell SKOV3 showed the most obvious changes after CGI-58 gene knockout. HCT-116 went through epithelia-mesenchymal transition shift(EMT) which promotes tumor cell invasion and metastasis. Meanwhile, SKOV3 went through a reverse process, mesenchymal- epithelial transition(MET), which inhibits tumor cell invasion and metastasis. It is an interesting phenomena that gene knockout of exactly the same gene led to opposite results in different cell lines. To further explore the mechanism of EMT-MET underlying lipid metabolism controlled by CGI-58 in cancer cells, also to determine the gene expression level of CGI-58 in ovarian cancer in clinical samples and its correlation with clinical pathological feature,this project was designed and validated.Objective: 1. To build up CGI-58 knockout ovarian cancer cell lines and metabolic change in CGI-58 knockout SKOV3 cells. 2. To observe biological effects of CGI-58 knockout in SKOV3 cells’ invasion and metastasis; to explore some key molecules and related cell signal pathway of EMT-MET; to clarify their function in CGI-58 knockout cancer cells. 3. To learn about the expression level of CGI-58 in clinical ovarian cancer tissues; to analyze the association between CGI-58 and clinical pathological feature. Material and Method 1. Material: Ovarian cancer cell line SKOV3 and OVCAR3, Colon cancer cell line HCT-116(including p53-null and p53-WT), ovarian cancer tissue from patients(after surgery), CGI-58 sh RNA plasmid, β-catenin overexpression plasmid and p53 overexpression plasmid; reagent for lentivirus compackaging, cell culture, immunohistochemiststry/immunofluroecent staining, Western Blot, and biochemistry. 2. Method: The cell lines which stably expressed CGI-58 sh RNA, β-catenin and p53 overexpression plasmid were set up through transfection and drug-selection; protein level and location analysis was done by Western Blot and immunofluorecent staining; lipid droplet and mitochondria activity were observed after immunofluorecent staining; the assay of triglyceride, free glycerol and ROS was completed by optical-density method, the growth and proliferation assays were done by CCK8 and clone forming experiments; the invasion and metastasis assays were done by scratch and transwell experiment; lipolysis was initialed by hormone, modified Lowry method and optical density method were applied in protein and triglyceride/glycerol level detection; tissue chips were applied to immunohistochemical staining and scored by pathologist, the final score was summed by scores of area and density.Result: 1. The SKOV3 cells had an abnormal storage of lipid droplets(LD) in cytoplasma after their CGI-58 gene silencing. There were obviously green fluorescent spots observed after BODIPY staining. Triglyceride assay result showed that these CGI-58 knockdown cells had a higher level of triglyceride than parental and control cells’. The result of lipolysis assayindicated HSL still maintained its normal function. It would work well under the stimulation of exogenous hormone. SKOV3 cells were active on lipid anabolism. The cells would quickly restore level of triglyceride from non-lipid material. 2. The Western Blot result of metabolic enzymes showed: In CGI-58 knockdown SKOV3 cells, AMPKα, the enzyme which control the ratio of ATP/AMP, was raised in phosphorylation form; ACC which control fatty acid synthase and AKT which control oxidative phosphorylation, went through no change in phosphorylation form; phos-GSK3β, responsible for inhibition of glycogen synthesis, was decreased. Mitochondrion staining showed mitochondrial respiratory and oxidative function was weakened in CGI-58 knockout SKOV3 cells. The slope of OD-time curve in ROS assay showed a higher trend in CGI-58 knockout cell comparing to control cells’, indicated that there were more reactive oxygen species produced in CGI-58 knockout cells than in control cells. 3. After CGI-58 silencing, SKOV3 cells had a lower speed of growth, which was proved by growth curve in CCK8 experiment. And its proliferation was also inhibited proved by clone forming experiment. There was a massive reduction in clone area of knockout cells comparing to control cells, despite same number of cells and same time of culture. 4. Morphological and Western Blot results showed: in CGI-58 knockout SKOV3 cells, epithelial markers E-cad increased level, meanwhile mesenchymal marker N-cad, vimentin and slug decreased level. The cell shape was transited from mesenchymal cell to epithelial cell. The results of scratch and transwell experiments showed CGI-58 knockout SKOV3 cells had lower capacity of invasion and metastasis. 5. The autophagy experiment result showed CGI-58 knockout SKOV3 cells had higher level of LC3, which means higher autophagy level comparing to control cells. An inhibitor of autophagy, 3-MA was administered in culture media, but no change was observed in E-cad after it inhibited the level of LC3 and autophagy. 6. In ovarian cancer cells, OVCAR3 that was p53-expressed showed a different E-cad shift pattern after CGI-58 knockout, comparing to SKOV3 that was p53-null. Colon cancer cell HCT-116, which went through EMT shift after CGI-58 knockdown, was silenced wild type p53 expression, after that, the E-cad level of CGI-58 knockout cells remained as same as the control cells. SKOV3 cell, which was p53-null and went through MET shift after CGI-58 knockout, was restored wild type p53 expression. E-cad level of knockout cells wasreduced obviously after p53 was induced, comparing to the simply knockout cells. 7. CGI-58 knockout SKOV3 cells had a reduction in Wnt-5a/b signal and β-catenin level. β-catenin constitutive active plasmid was transduced into both control cells and knockout cells through lentivirus vector. After it was stably expressed, WB result showed that there was a decreased level in E-cad and increased level in N-cad. And CGI-58 knockout cell’s shape was transitted from epithelial cell to mesenchymal cell. 8. Immunohistochemistry staining and scoring result in clinical ovarian cancer tissue showed CGI-58 positive(include strongly) expression in ovarian cancer tissue was up to 85.4%. The expression level was only obviously associated with tumor grade, not with age, lymph node invasion, instant metastasis and clinical stage.Conclusion: 1. CGI-58 played an important role in lipid catabolism in SKOV3 cells: CGI-58 knockout would bring abnormal storage of triglyceride, shortage of ATP, reduction in respiration and oxidation function of mitochondrion, and lipid reprogramming in cells. This would change metabolism pattern of SKOV3 cells. 2. The metabolism reprogramming brought by CGI-58 knockout in SKOV3 cells resulted inhibition of growth and proliferation and rising of autophagy The most characteristic change was reduced capacity of invasion and metastasis. CGI-58 knockout cells had a MET shift in morphology. 3. The EMT-MET shift after CGI-58 knockout in cancer cell lines depended upon p53 status. p53 status would decide the cell shape shift direction to EMT or MET, which was brought by CGI-58 knockout. 4. Among ovarian cancer tissues, expression level of CGI-58 was associated with the depth and range of ovarian cancer infiltration. It could reflect capacity of cancer cells’ local invasion. CGI-58 would potentially be a marker, which could indicate the prognosis of ovarian cancer patients.