Mechanism Of KLF-9 Regulating The Stem Cell-Like Phenotype Of Ovarian Cancer Ascites-Derived Multicellular Spheroids

Ovarian cancer is the second leading cause of the female reproductive system cancer death.Direct spread and implantation metastasis is the most common type of metastasis in ovarian cancer.Tumor cells shed from the primary tumor,survive in ascites and further form implantation lesions,which are the main processes of peritoneal metastasis in ovarian cancer.The exfoliated tumor cells adhere to each other and aggregate to form spherical multicellular spheroids or multicellular clusters(MCSs/MCAs)while self-renewing.Ovarian cancer MCSs are the main seed cells for intraperitoneal metastasis,however,their biological properties and survival mechanisms remain unclear.In this study,we found that ovarian cancer MCSs exhibited cancer stem-like characteristics(CSLC),including expression of cancer stem cell-related markers,self-renewal ability,chemoresistance and high tumorigenicity in nude mice.Mechanistic studies have shown that Krüppel-like factor 9(KLF9)is low expressed in ovarian cancer ascites-derived MCSs and ovarian cancer cell line-derived MCSs.KLF9 can directly bind to the Notchl promoter region and regulate Slug expression in a N1ICD-CSL-dependent manner,thus involved in the regulation of ovarian cancer stem cell-like phenotype.Clinically,KLF9 expression correlates with histological grade in epithelial ovarian cancer,and loss of KLF9 predicts poor ovarian cancer outcomes.Our main research is divided into 3 parts:Part Ⅰ:Human ovarian cancer ascites-derived MCSs exhibit a stem cell like phenotype.Part Ⅱ:The expression of KLFs in ovarian cancer MCSs and clinical,biological significance of KLF9.Part Ⅲ:N1ICD-CSL/Slug involves in KLF9-mediated modulation of stemness in ovarian cancer MCSs.Part Ⅰ Human ovarian cancer ascites-derived MCSs exhibit a stem cell like phenotype Objective:To identify and characterise human ovarian cancer ascites-derived MCSs.To investigate CSC-related phenotype of MCSs compared with primary tumor.Methods:1.MCAs in ascites/peritoneal washings specimens of EOC patients in the Department of Obstetrics and Gynecology,Shandong University Qilu hospital were isolated and the presence of epithelial tumor cells of ascites-derived MCSs was vadified by flow cytometry and immunofluorescence detection of EpCAM.2.Transwell assays were performed to determine cell migration and invasion ability of MCSs and primary tumor cells.3.Flow cytometry was used to detect the proportion of CD44+ and CD 117+ cells in MCSs,primary tumor and metastases cells.4.Sphere-forming assay,cell viability assay and limiting dilution assay were conducted to determine CSC-related phenotype of MCSs compared with primary tumor cells.5.Quantitative real-time polymerase chain and western blot were performed to detect mRNA and protein level of stemness and EMT-related markers in the ascites-derived MCSs and primary tumor cells.Results:1.Light microscopy shows ascites-derived MCSs in clusters of multicellular aggregates,30-200um in diameter,with a spherical shape.Flow analysis shows that MCSs are negative for the leukocyte marker CD45 and positive for the epithelial tumour marker EPCAM.Immunofluorescence showed that peritoneal MCSs expressed EpCAM,further indicating that the cancer cells shed from the peritoneal cavity of ovarian cancer patients were epithelial in origin.2.The transwell assay confirmed the higher cell migration and invasion capabilities of MCSs cells compared with primary tumor cells.Cell viability assay showed that MCSs cells exhibited stronger resistance to drugs during cisplatin treatment than primary tumor cells.3.To investigate cancer stem cell phenotype of MCSs,we performed sphere formation assay,limiting dilution assasy,flow cytometry and detected stemness-related markers.Sphere formation assay showed that peritoneal MCSs cells formed large-sized and denser tumor spheres cultured in a serum-free,semi-solid medium compared with primary tumor cells.Limiting dilution assay revealed that MCSs cells possess stronger self-renewal capacity.QRT-PCR showed that MCSs cells expressed a higher level of stemness-related genes,including Nanog,SOX2 and OCT-4.In addition,flow cytometry illustrated a higher proportion of CD44+and CD117+ cells in MCSs than primary tumor and metastases.4.Since EMT plays important role in metastatic process,we believed ascites tumor cells underwent EMT response during aggregation process of MCSs acquired a mesenchymal-like phenotype.To investigate it,we detected the level of EMT-related markers,including Snail,Slug,Twist,Zebl and Zeb2.The results showed that Snail,Slug and Twist were significantly up-regulated in MCSs and increased during sphere forming process of primary tumor cells.In addition,SB-431542(a TGF-β receptor inhibitor)treatment resulted in less cohesive and smaller spheroids than DMSO group by suppressing EMT phenotype as evidenced by a significant down-regulation in EMT-related markers mRNA expression,which indicated that EMT is involved in MCSs formation and cancer stem cell genesis.Conclusions:1.Ovarian cancer ascites-derived MCSs showed significantly enhanced migration and invasion ability,drug resistance,self-renewal ability and up-regulated expression of relevant tumour stem cell markers compared to primary tumour cells,exhibiting stem cell-like biological characteristics.2.EMT participates in cancer stem cell genesis and human OC ascites-derived MCSs exhibit a mesenchymal-like phenotype.Part II:The expression of KLFs in ovarian cancer MCSs and clinical,biological significance of KLF9Objective:To investigate the expression of KLFs in ascites-derived MCSs,explore the alteration of KLF9 in the process of sphere formation,re-differentiation and evaluate the correlation between KLF9 expression and clinico-pathological features of EOC patients.To investigate the role of KLF9 in regulating stem-like phenotype of human ovarian cancer cell line.Methods:1.Quantitative real-time polymerase chain was performed to detect mRNA level of KLFs family members in the ascites-derived MCSs.2.QRT-PCR and western blotting were used to measure the expression of KLF9 in MCSs,primary tumor cells and metastases.3.Gene datasets of GSE28799 from GEO database were analyzed to explore expression of KLF9 in ovarian cancer cell line and cell line-derived spheroids.4.GSE33874 datasets were used to analyse KLF9 expression in side population(SP)compared with main population(MP)isolated from EOC ascites tumor cells.5.For sphere formation assay,we induced the EOC cell lines into spheroids in 3D serum-free culture condition.For re-differentiation assay,we cultured spheroids in complete medium.QRT-PCR and western blot were performed to detect alteration of KLF9 and stemness-related markers.CD117+/CD44+and CD117-/CD44-subset cells from ascites-derived MCSs were isolated by use of FACSAria II flow cytometer.6.KLF9 protein in EOC tissues collected from the department of obstetrics and gynecology of Qilu Hospital was detected by immunohistochemistry,and the relationship between KLF9 expression and clinical characteristics was analyzed.7.The siRNA and plasmids purchased from Genechem(Shanghai,China)were transfected into ovarian cancer cell lines to down-regulate or up-regulate KLF9 expression following the manufacturer’s protocols.The cells were cultured in serum-free,semi-solid medium to induce spheroids.8.QRT-PCR and western blot were used to measure the level of KLF9 and stemness-related markers.Flow cytometry was used to detect the proportion of CD44+/CD24-cells in cell line-derived spheroids.Sphere-forming assay,cell viability assay and apoptotic assay were conducted to determine CSC-related phenotype of EOC cells.9.Different gradient xenograft tumorigenicity experiments were used to further investigate the effect of KLF9 in ovarian cancer tumorigenesis in vivo.Results:1.To explore the role of KLFs family in the maintenance of CSC properties in ovarian cancer MCSs,we detected the mRNA level of nine of the KLFs,including KLF4,KLF5,KLF6,KLF8,KLF9,KLF10,KLF11,KLF13,KLF16,which were able to generate detectable PCR products in ovarian cancer specimen and observed that KLF9 was significantly down-regulated in ascites-derived MCSs.This MCSs-specific expression pattern of KLF9 was further observed at mRNA and protein level.Additionally,we did not find significant difference in the level of KLF9 in metastatic lesions compared with the primary tumor.2.Analysis of GSE28799 datasets indicated that KLF9 was down-regulated in OVCAR-3 spheroids which derived from parental EOC cell line OVCAR-3.The mRNA and protein expression of stemness-related markers exhibited an increasing trend during the process of spheroids formation.Conversely,KLF9 expression decreased.In addition,we observed the opposite phenomenon during the process of re-differentiation.3.Analysis of GSE33874 datasets indicated that KLF9 was down-regulated in side population(SP)compared with main population(MP)isolated from ascites of EOC patients.Then,we isolated CD117+CD44+and CD117-CD44-subgroup from MCSs and found that KLF9 maintained low expression levels in CD117+CD44+cells.4.Immunohistochemical results showed that KLF9 expression was correlated with histological grade.No statistical significance was found in the correlation between KLF9 expression and other parameters including age,FIGO stage,histological type,lymph node metastasis,residual disease,clinical response and platinum resistance.Kaplan-Meier survival analysis indicated that the patients with low KLF9 expression had a poor prognosis.5.QRT-PCR and western blot analysis showed that KLF9 over-expression in SK-spheres decreased the level of stemness-related markers,including Nanog and SOX2.In opposite,knockdown of KLF9 in HO-spheres increased Nanog and SOX2 expression.Flow cytometry analysis indicated that KLF9 over-expression decreased the proportion of CD44+/CD24subgroup in SK-spheres,while KLF9 knockdown increased proportion of CD44+/CD24subgroup in HO-spheres.6.Serum-free suspension culture results showed that over-expression of KLF9 reduced both the number and size of tumour spheres,which indicated the weakened sphere formation ability and self-renewal of cells in KLF9-OE group.KLF9-KD group showed an opposite phenomenon.7.Cell viability assay and Annexin V/PI double staining apoptotic assay demonstrated that KLF9-OE SK-spheres showed lower cell survival during CDDP treatment than negative control group,while KLF9-KD HO-spheres group showed an opposite results.8.The effect of KLF9 on tumorigenesis of EOC-spheres cells in mice was detected by different gradient xenograft tumorigenicity experiments.Compared with control group,KLF9-OE SK-spheres(1*105 cells)exhibited significantly reduced tumor size and KLF9-OE SK-spheres(1*104 cells)generated significantly reduced tumor number and size.Immunohistochemical analysis showed a decrease of CD117 expression after KLF9 over-expression,which represents the inhibitory effect of KLF9 on EOC stemness.Conclusions:1.KLF9 is significantly down-regulated in ascites-derived MCSs at mRNA and protein level,which imply an important role of KLF9 in ovarian cancer stem cell.2.Stemness-related markers increase during process of spheroids formation,while decrease during re-differentiation.The expression of KLF9 decreases during process of spheroids formation,while increase during re-differentiation.3.KLF9 maintained lower expression level in CD117+CD44+cells compared with CD117-CD44-cells isolated from EOC ascites-derived MCSs.4.KLF9 expression is correlated with histological grade of EOC tissues and low expression of KLF9 predicts a poor prognosis.5.KLF9 deletion increases stemness-related markers,proportion of CD44+/CD24-cells,strengthens sphere forming capacity and drug resistance of EOC spheroids.KLF9 is involved in regulating stem-like properties in vitro.6.KLF9 inhibits xenograft tumorigenesis ability of EOC spheroids in vivo.Part Ⅲ:N1ICD-CSL/Slug involves in KLF9-mediated modulation of stemness in ovarian cancer MCSs.Objective:To explore the downstream target of KLF9 and the regulatory mechanisms.Methods:1.QRT-PCR and western blotting analysis were used to measure the mRNA and protein level of EMT-related markers.2.Expression of KLF9 and Slug in cell line(monolayer)and cell-derived spheroids(spheres)were detected by western blotting.3.Western blotting,sphere formation assay,flow cytometry,cell viability assay,subcutaneous xenografts and peritoneal metastasis mice experiments were performed to conduct stem-like phenotype rescue assay.4.ChIP(Chromatin immunoprecipitation)assay and Luciferase activity assay were used to explore direct function mechanisms between genes.5.Immunohistochemistry,qRT-PCR and western blotting analysis were used to investigate the co-expression of KLF9 and downstream target.Results:1.After KLF9 over-expression,mRNA and protein levels of the epithelial markers E-cadherin was significantly up-regulated and the levels of the mesenchymal markers,N-cadherin and vimentin,were down-regulated.Among EMT-related TFs which were up-regulated in MCSs,Slug was significantly down-regulated after KLF9 over-expression.Western blotting assay indicated a higher level of slug associated with a lower expression of KLF9 in cell-derived spheroids,as compared to those in parental cells.2.Rescue experiments of Slug in KLF9-mediated regulation of stem-like phenotype in EOC cells showed the KLF9-OE induced down-regulation of stemness-related markers,decreased CD44+/CD24-proportion in spheroids,weakened sphere forming capacity and reduced resistance to cisplatin were reversed,at least partially,by slug over-expression.Additionally,the KLF9-KD induced strengthened stemness were reversed by slug knockdown in vitro and in vivo.3.Notchl was predicted to be a direct downstream target of KLF9 by analyzing KLF9 ChIP-seq data from human ENCODE database.Luciferase activity assay and ChIP assay confirmed that KLF9 decreased the transcriptional expression of Notchl by binding to Notchl promoter directly.Sphere formation assay,flow cytometry and peritoneal metastasis mice experiments revealed that Notchl is involved in KLF9-mediated inhibition of OCSC development.4.Western blotting assay indicated that KLF9 regulates expression of Slug in a Notchl-dependent manner.Western blotting and qRT-PCR showed a significant negative correlation between the protein,mRNA level of KLF9 and Notchl,Slug in MCSs samples.Si-RNA assay confirmed that Notchl regulates expression of Slug via CSL rather than Hesl or Hey 1.Luciferase activity assay further validated that CSL-binding site acts as a Notchl-response element in Slug promoter and KLF9 modulates Slug in a Notch1-CSL dependent manner.Conclusions:1.Slug plays crucial role in KLF9-mediated regulation of stem-like phenotype in ovarian cancer cells.2.Notchl is the direct downstream molecule of KLF9 in ovarian cancer cells.3.KLF9 modulates the expression of Slug via CSL-Notchl complex.