S100B Mediates Stemness Of Ovarian Cancer Stem Cells

BackgroundOvarian cancer is the most common and lethal gynecological cancer disease in the world.Despite the current treatment in the treatment of ovarian cancer has achieved certain therapeutic effect,the 5 year survival rate of ovarian cancer patients is still less than 30%.It has been found that easy to relapse and metastasis determines the poor prognosis of patients with ovarian cancer.Recent studies have shown that there is a small part of cells harboring stem cell properties in ovarian cancer tissues,named ovarian cancer stem cells(OCSCs).Moreover,OCSCs are closely related to the occurrence,development,metastasis and prognosis of ovarian cancer.Therefore,it is important to clarify the regulating mechanisms of ovarian cancer stem cells for the development of new treatment strategies and improvement of the prognosis of patients.Cancer stem cells exist in a unique microenvironment just like adult stem cells,called tumor stem cell niche.Tumor stem cell niche consists of various kinds of cells,including tumor cells,vascular endothelial cells,fibroblasts and many kinds of immune cells.Various kinds of cells in the stem cell niche can regulate the self-renewal,multi lineage differentiation and drug resistance of cancer stem cells by secreting various cytokines or chemokines.A large number of studies have found that ovarian cancer stem cells are also regulated by many kinds of inflammatory factors(such as IL-17),which are derived from stem cell nests.However,there is still a lack of in-depth and systematic research on the regulation mechanism of ovarian cancer stem cells.In our previous study,we have successfully isolated CD133 positive ovarian cancer stem cells and identified their stem cell properties,including self-renewal,multi-lineage differentiation and tumorigenicity.In order to fully understand the self regulatory mechanisms of ovarian cancer stem cells,we have acompared the expression of inflammatory factors between CD133+ ovarian cancer stem cells and CD133-ovarian cancer cells by PCR-array.We found that in addition to the reported IL-4,IL-8 and LIF and other factors,there are still no reports of inflammatory cytokines was also highly expressed in ovarian cancer stem cells.Among them,S100 B is the most differential expression of the inflammatory factors in these two kinds of cells,which has attracted our attention.The S100 family consists of 21 Ca2+ binding proteins with EF hand structure,and S100 B is one of the members of the family.The abnormal expression of S100 B in nervous system diseases and many kinds of tumors were reported.However,its role in cancer stem cells is not clear.Previous studies found that the extracellular S100 B could activate the NF-? B signa ling pathway in small nerve glial cells and enhance the expression of pro-inflammatory cytokine COX2;and intracellular S100 B could resist its phosphorylation through the interaction with p53 protein,,then affect p53 function.NF-? B signaling pathway and p53 protein have been reported to play important roles in maintaining the self-renewal of cancer stem cells.Therefore,this study further clarified the role of S100 B in the regulation of ovarian cancer stem cells and the underlyding molecular mechanisms based on our previous studies.Objectives1.To detect the expression of S100 B in ovarian cancer tissues and ovarian cancer stem cells.2.To study the roles of S100 B in regulation of stemness of ovarian cancer stem cells.3.To elucidate the underlying mechanisms of S100 B regulation of stemness of ovarian cancer stem cells.Methods1.To detect the expression of S100 B in ovarian cancer tissues and ovarian cancer stem cells.Firstly,immunohistochemistry(IHC)was used to determine the expression of S100 B in human ovarian cancer and non-tumor tissues,and the expression of S100 B in human ovarian cancer tissues and non tumor tissues was also analyzed with GEO database.Then,the correlations between the expression of S100 B in ovarian carcinoma and tumor stages,degree of tumor differentiation and patients' survival were analyzed in GEO database.At last,the expression of S100 B in ovarian cancer stem cells and non stem cells derived from cell lines and primary tumors were determined by Real-time PCR,Western blot and imm uno fluoresce nce staining.And the co-expression of S100 B and CD133 was detected in the primary ovari an tumors by immunofluorescence staining.2.To study the roles of S100 B in regulation of stemness of ovarian cancer stem cells.The S100 B expression in ovarian cancer stem cells derived from cell lines and primary tumor tissues was knocked down by transfecting S100 B sh RNA lentivirus,and the expression of S100 B was detected by Real time PCR and Western blot.In vitro sphere-forming assay was used to detect the effects of S100 B knockdown after self-renewal capacity of ovarian cancer stem cell;xenografted tumor model in nude mice was used to determine the effect of S100 B knockdown on tumorigenicity of ovarian cancer stem cells in vivo,and flow cytometry was used to detect the proportion of CD133 positive cells in xenografted tumors.Western blot and immunofluorescence staining were performed to detect the effects of S100 B knockdown on the expression of stemness markers,including Naong,Oct4 and Sox2 in ovarian cancer stem cells and the xenografted tumors.The S100 B expression in A2780 cells was over-expressed by transfecting S100 B lentivirus,and the expression of S100 B was detected by Western blot.In vitro sphere-forming assay was used to detect the effects of S100 B overexpression on self-renewal capacity of ovarian cancer cells,and flow cytometry was used to detect the proportion of CD133 positive cells in cell lines;xenografted tumor model in nude mice was used to determine the effect of S100 B overexpression on tumorigenicity of A2780 cells in vivo.Western blot and immunofluorescence staining were performed to detect the effects of S100 B overexpression on the expression of stemness markers,including Naong,Oct4 and Sox2 in A2780 cells and the xenografted tumors.3.To elucidate the underlying mechanisms of S100 B regulation of stemness of ovarian cancer stem cells.Flow cytometry and immunofluorescence staining were used to detect expression of S100 B receptor,advanced glycation end product receptor(RAGE)in ovarian cancer stem cells and non stem cells.After blocking S100B-RAGE signaling pathway with RAGE blocking antibody,the self-renewal capacity of ovarian cancer stem cells was determined by sphere-forming assay in vitro.The activation of NF-kappa B signaling pathway was determined by Western blot in ovarian cancer stem cells with S100 B knockdown.The effects of S100 B knockdown on expression of p53 and phosphorylated-p53 were determined by Western blot in ovarian cancer stem cells derived from A2780 cells and xenografted tumors.And the effects of S100 B overexpression on expression of p53 and phosphorylated-p53 were determined by Western blot in A2780 cells.Moreover,the expression of S100 B,p53 and phosphorylated-p53 was also determined in primary tumor tissue by Western blot.And then p53 protein was knocked down in ovarian cancer stem cells by transfected them with p53 sh RNA lentiviral vector.The expression of p53,phosphorylated-p53 and stemness markers in ovarian cancer stem cells with or without S100 B knockdown was determined by western blot.At last,the effects of p53 knockdown on self-renewal capacity of ovarian cancer stem cells were determined by sphere-forming assay in vitro.Results1.S100 B was highly expressed in ovarian cancer tissues and was correlated with tumor stage,degree of differentiation and the prognosis of the patients.The expression of S100 B was detected by immunohistochemistry in 10 human ovarian cancer tissues and adjacent non-tumor tissues.It was found that the expression of S100 B was significantly higher than that in adjacent tissues.Through analyzing the GEO database,we also found that the expression of S100 B in ovarian cancer tissues was significantly higher than that in non-tumor tissue.Moreover,we also found that the expression of S100 B was negatively correlated with tumor stage and the prognosis of the patients.Then we examined the expression of S100 B in 15 ovarian cancer tissues with different degrees of differentiation by Real-time PCR and analyzed the correlations between S100 B expression and degree of differentiation.We found that S100 B expression was lower in ovarian cancer tissues with well differentiation,while higher in ones with poor differentiation.2.S100 B is preferentially expressed in ovarian cancer stem cells.Real time PCR,Western blot and immunofluorescence staining were used to detect S100 B expression in ovarian cancer stem cells derived A2780 cell lines.The results showed that both m RNA and protein of S100 B was significantly higher in ovarian cancer stem cells than that in non stem cells.Then CD133+ ovarian cancer stem cells and CD133-non stem cells were isolated by Macs from human primary ovarian carcinoma tissue,and the expression of S100 B was detected by Real-time PCR and Western blot.The results showed that S100 B expression was alos significantly higher in ovarian cancer stem cells than that of in non stem cells derived from primary tumor tissues.Finally,we performed immunofluorescence staining to detect the co-expression of S100 B and CD133 in human ovarian cancer specimens in situ.The results showed that S100 B and CD133 were co-expressed.These data further indicate that S100 B was mainly expressed in cancer stem cells of ovarian cancer tissue.3.S100 B knockdown significantly inhibited the self-renewal,tumorigenicity and expression of stemness markers of ovarian cancer stem cells.In order to understand the roles of S100 B in ovarian cancer stem cells,we knocked down the S100 B expression in ovarian cancer stem cells derived from cell lines and primary tumor tissues by tranfecting them with S100 B sh RNA lentiviral vectors.The results of Real time PCR and Western blot showed that S100 B expression was significantly decreased after S100 B sh RNA transfection.Then we determined the effects of S100 B knockdown on the self-renewal ability,tumorigenicity and the expression of stemness markers of ovarian cancer stem cells.The sphere-forming assay revealed that S100 B knockdown significantly decreased the number and size of spheres of ovarian cancer stem cells.And we also found that S100 B knockdown significantly inhibit the tumorigenicity of ovarian cancer stem cells using xenografted tumor models in nude mice,and the results of flow cytometry revealed that the proportion of CD133 + cells was also significantly decreased when S100 B was knocked down.Western blot and immunofluorescence showed that S100 B knockdown also decreased the expression of stemness markers,including Nanog,Oct4 and Sox2 in ovarian cancer stem cells and xenografted tumors.4.S100 B overexpression could endow ovarian cancer cells with stem cell properties.In order to further understand the roles of S100 B in the regulation of ovarian cancer stem cells,S100 B was overexpressed in ovarian cancer cell line A2780 by transfecting S100 B lentiviral vector.The overexpression of S100 B was identified by western blot.Then we examined the effects of S100 B overexpression on the sphere-forming,tumorigenicity and expression of stemness markers.Sphere-forming assay revealed that S100 B overexpression enhanced the self-renewal of A2780 cells,and the percentage of CD133+ cells was significantly increased after S100 B overexpression.We found S100 B overexpression also significantly increased the tumorigenicity of A2780 cells.Western blot and immun ofluore scence revealed that S100 B overexpression in A2780 cells significantly upregulated the expression of stemness markers,including Nanog,Oct4 and Sox2.5.S100 B mediates stemness of ovarian cancer stem cells through inhibiting p53 activity.In order to clarify the molecular mechanisms of S100 B in regulation of ovarian cancer stem cells,we first detected the expression of S100 B receptor-RAGE in ovarian cancer stem cells.The results of flow cytometry and immunofluorescence showed that ovarian cancer stem cells almost expressed RAGE.Then we detected the effects of RAGE blockade on ovarian cancer stem cell self-renewal by antibody blocking,and the results showed that RAGE blocking did not affect the formation of ovarian cancer stem cells.In addition,we also detected the effects of S100 B knockdown on S100B-RAGE-NF-kappa B signaling pathway,and western blot found that the activation of NF-kappa B signaling pathway in ovarian cancer stem cells was not changed after S100 B knockdown.These results suggest that the regulation of S100 B on ovarian cancer stem cells is independent on the S100B-RAGE-NF-kappa B signaling pathway.In order to further clarify the molecular mechanisms of S100 B regulation stemness of ovarian cancer stem cells,we detected the effects of S100 B in the regulation of p53 protein in ovarian cancer stem cells.Western blot showed that S100 B knockdown could significantly increase the expression of p53 and phosphorylated p53 in ovarian cancer stem cells,while S100 B overexpression significantly inhibited the expression of p53 and phosphorylated p53 in ovarian cancer cell line A2780.We also found that the expression of S100 B was negatively correlated with the expression of p53 and phosphorylated p53 in primary ovarian cancer tissues.Then we knocked down p53 expression in ovarian cancer stem cells through transfecting p53 sh RNA lentiviral vector to further detect whether the S100 B regulation stemness of ovarian cancer stem cells was dependent on the p53 inactivation.Western blot showed that p53 knockdown could partially restore the expression of stemness markers in ovarian cancer stem cells induced by S100 B knockdown.In addition,we also found that the p53 knockdown could partially restore the self renewal ability of ovarian cancer stem cells induced by S100 B knockdown.These results indicate that the regulation of 100 B on ovarian cancer stem cells is dependent on the inactivation of p53.ConclusionIn this study,we revealed the important roles of the S100B-p53 pathway in regulation stemness of ovarian cancer stem cells from a self-regulation perspective.We found that S100 B was highly expressed in ovarian cancer tissues,especially in ovarian cancer stem cells.The expression of S100 B was positively correlated with the expression of tumor stem cell markers(CD133,Nanog and Oct-4).S100 B knockdown significantly decreased the self-renewal ability,tumorigenicity and expression of stem cell markers of ovarian cancer stem cells.And S100 B overexpression could induce ovarian cancer cells with stem cell properties.S100 B mediates the stemness of ovarian cancer stem cells was independent on the activation of the RAGE-NF-kappa B pathway,but dependent on p53 inhibition.In this study,we further clarify the roles and mechanisms of S100 B in the self-regulation of ovarian cancer stem cells,and provide a new molecular target for the treatment of ovarian cancer stem cells.