H, Protein level of KLF9 in CD117+CD44+ and CD117?CD44? subsets of ascites\MCSs detected by western blot 3.3. MCSs. Functional experiments demonstrated that KLF9 negatively modulated stem\like properties in OC cells. Mechanistic studies revealed that KLF9 reduced the transcriptional expression of Notch1 by directly binding to the Notch1 promoter, thereby inhibiting the function of slug in a CSL\dependent manner. Clinically, expression of KLF9 was associated with histological grade and loss of KLF9 predicts poor prognosis in OC. strong class=”kwd-title” Keywords: KLF9, multicellular spheroids, Notch1, ovarian cancer stem cells, slug Abstract Ascites\derived MCSs exhibited cancer stem cell (CSC) properties concomitant with enhanced malignancy, induced EMT, and low KLF9 expression, compared with PTCs. KLF9 reduced the transcriptional expression of Notch1 by directly binding to the Notch1 promoter, thereby inhibiting the function of slug in a Urocanic acid CSL\dependent manner. AbbreviationsCCK\8Cell Counting Kit\8ChIPchromatin immunoprecipitationCSLCBF1/Su (H)/Lag\1 (also known as RBP\Jkappa)EMTepithelial mesenchymal transitionEpCAMepithelial cell adhesion moleculeIRSimmunoreactive scoreKDknocked downKLF9Krppel\like factor 9MCSs/MCAsmulticellular spheroids/aggregatesMPmain populationN1ICDNotch1 intracellular domainOCovarian cancerOCSCovarian cancer stem cellsOEover\expressingPTCsprimary tumor cellsSFMserum\free mediumSPside populationTFstranscription factorsTSStranscription start site 1.?INTRODUCTION Metastasis of OC is the most common cause of disease\associated mortality. In the majority of cases, the disease has spread beyond ovaries at the time of diagnosis.1 Metastasis is a dynamic, multistep and complex process, including escaping from primary tumor, spreading in the systemic circulation, extravasation at distant tissues, and organ seeding.2 Among diverse metastatic routes, peritoneal implantation is the most common mode of metastasis for OC.3 At the cellular level, cancer cells exfoliated from the primary site to a fluid\filled peritoneal cavity where they exist in suspension as single cells or MCSs. OC MCSs contribute to the production and accumulation of large amounts of malignant ascites and are the major seeds of peritoneal metastasis.4 Therefore, improved understanding of properties, underlying molecular mechanisms of the cellular biology of OC MCSs will shed light on novel therapeutic options. Cancer stem cells share several biological characteristics with normal stem cells, including self\renewal, asymmetric cell division, and resistance to apoptosis induced by an anchorage\independent manner.5 In addition, a defining phenotype restricted to CSCs is their ability for efficient tumorigenesis, drug resistance, and the acquisition of mesenchymal traits through EMT.6, 7, 8 Based on this property, CSCs are also commonly called tumor\initiating cells which were defined as the ability of a few cells to form a xenograft representative of the parental neoplasm.5 CSCs frequently express a quiescent state that makes them resistant to chemotherapy and radiotherapy that target actively proliferating cells.9, 10 EMT endows cell plasticity, and enables the generation of CSCs at different steps of the metastatic process.3 Regarding CSC in OC, OC cells are able to switch between epithelial and mesenchymal states during intraperitoneal implantation. The presence of peritoneal MCSs that can survive and proliferate even in the absence of adhesion to a substrate supports the hypothesis that the disease is driven and sustained by CSC and EMT is involved in the process. TFs have been documented for the occurrence and development of a variety of cancers.11 The Krppel\like factor family of TFs consists of 17 conserved proteins containing zinc finger and modulates diverse biologic processes, including cell differentiation, apoptosis, EMT, and stemness.12, 13, 14 Krppel\like factor 9 (KLF9) is a TF that binds to GC box elements located in the promoter and has been found to be downregulated in colorectal cancer and endometrial carcinoma.15, 16, 17 In the last few years, KLF9 has been reported to be associated with gastric cancer, breast cancer, hepatocellular carcinoma, and prostate cancer.18, 19, 20, 21 Rabbit Polyclonal to CtBP1 In addition, KLF9 was downregulated in glioblastoma\derived spheroids and inhibited glioblastoma stemness.22, 23, 24 However, the role of KLF9 in ovarian CSCs (OCSCs) remains undefined. Here we use ascites\derived MCSs and spheres induced from human OC cells to detect the expression and function of KLF9 in tumor\initiating CSCs. We found that KLF9 was differentially expressed between MCSs and PTCs, downregulated during the spheroid\formation process of OC cells. Functional assays showed that KLF9 Urocanic acid negatively modulates Urocanic acid stem cell\like properties. In the mechanism, KLF9 regulates OCSC via CSL\dependent Notch1/slug signaling, which may inspire new insights on clinical management of OC. 2.?MATERIALS AND METHODS 2.1. Patients and tissue samples Detailed in Appendix?S1. 2.2. Cell culture, sphere\forming, and re\differentiation assay Detailed in Appendix?S1. 2.3..
H, Protein level of KLF9 in CD117+CD44+ and CD117?CD44? subsets of ascites\MCSs detected by western blot 3