Interleukin-17Produced By Tumor Microenvironment Promotes Self-renewal Of CD133~+Cancer Stem-like Cells In Ovarian Cancer

BackgroundOvarian cancer is the most lethal form of gynecological cancer. Clinical characteristics ofovarian carcinoma include no particular symptoms in the early stage, high rate of diseaserecurrence and easily metastasize to distinct sites. more and more evidences reveals that onlya minority of cells within the tumor have the properties of stem cell，this rare fraction of cellstermed cancer stem cells（CSCs）or cancer stem-like cells（CSLCs）which are thought to beresponsible for cancer initiation, recurrence and metastasis. Correspondingly, ovarian cancerstem cells (OCSCs) are also importance for the ovarian tumorigenesis and progression.Therefore, OCSCs could be a novel therapeutic strategy for the ovarian cancer.Beside higher invasive potential, resistant to treatment with a variety ofchemotherapeutics than normal cancer cells, the essential characteristic of the CSCs isself-renewal. Cancer initiation, recurrence and metastasis depend on the self-renewalcapability of CSCs. So self-renewal is a key mechanism of CSCs research.CSCs self-renewal was regulated by intrinsic cellular pathways as well as extrinsicsignals generated by the tumor microenvironment. Cancer-related inflammation is thehallmark of tumor microenvironment. However, the effect of Cancer-related inflammation onOCSCs remains to be detailed explored. The thesis discusses these.Objectives1．Detect the expression of inflammation-associated factors and receptors in CD133+ovarian cancer stem-like cells（OCSLCs）.2．Define the function of IL-17in the self-renewal of CD133+OCSLCs.3．Clarify the mechanisms of IL-17on ovarian CD133+OCSLCs.Methods1．Detect the expression of inflammation-associated factors and receptors in CD133+OCSLCs. （1）Using the Human Inflammatory Response PCR Array to analyze the geneexpression of inflammation-associated factors and receptors genes between A2780derivedovarian CD133+OCSLCs and non-OCSLCs (CD133-A2780cells).（2）Verifying the expression of IL-17R in A2780cell line, A2780-derived ovarianCD133+OCSLCs and human fresh ovarian cancer tissue by flow cytometric analysis andimmunofluorescence assay.2．Evaluate the function of IL-17in the self-renewal of CD133+OCSLCs.（1）Detecting the IL-17producing cells in ovarian cancer by immunohistochemistry.（2）Studying the function of IL-17in the self-renewal of CD133+OCSLCs by Sphereformation assay and receptor inhibitor.（3）Studying the function of IL-17in the self-renewal of CD133+OCSLCs bytransfecting CD133+OCSLCs with IL-17-expressing Lentivirus and nude mousetumor-bearing assay.3．Investigate the mechanisms of IL-17on ovarian CD133+OCSLCs.（1）Screening and verifying the self-renewal related genes of IL-17on ovarian CD133+OCSLCs by Microarray and quantitative real-time PCR.（2）Finding the downstream signaling pathway of IL-17on ovarian CD133+OCSLCsby Signaling Net Array.（3）Verifying the NF-κB and p38MAPK signaling pathway in IL-17on ovarianCD133+OCSLCs by western-blot, immunofluorescence and antibody inhibitor assay.Results1．Expression of IL-17R on the ovarian CD133+OCSLCsWe first used the Human Inflammatory Response PCR Array to analyze the geneexpression of inflammation-associated genes between A2780derived ovarian CD133+OCSLCs and non-CSLCs(CD133-A2780cells),combing the biological effects of these gene，we decided to futher study the IL17R in A2780-derived ovarian CD133+OCSLCs.Flow cytometric and immunofluorescence assay revealed that the percentage of IL-17Rexpressing cells in the A2780derived ovarian CD133+OCSLCs was higher than non-CSLCs.primary ovarian CD133+OCSLCs also expressed IL-17R. This result point that IL-17interactwith CD133+OCSLCs has the possibility on material basis.2．IL-17promotes ovarian CD133+OCSLCs self-renewal IL-17R functions through binding with its ligand IL-17. Therefore, we first detectedIL-17producing cells in ovarian cancer using immunohistochemistry. The IL-17positive-staining cells were located mainly in the stroma of tumor section. Further confirmthat the majority of IL-17positive-staining cells were CD4+lymphocytes and CD68+macrophages. Moreover, we found that IL-17-producing cells locate in the niche of CD133+OCSLCs in ovarian cancer tissues. This result point that IL-17interact with CD133+OCSLCshas the possibility on space.Sphere formation assay conferred that the number of spheres and size of spheresignificantly increased by IL-17stimulation in vitro. In addition, the effect of rhIL-17wasblocked by an IL-17R neutralizing antibody.To further evaluate the role of IL-17on ovarian CD133+OCSLCs self-renewal in vivo,we get ectopic expression of IL-17in CD133+OCSLCs using lentivirus-mediatedtransduction to mimic IL-17in vivo effect on CD133+OCSLCs. Consistent with the treatmentof rhIL-17, IL-17-transduced ovarian CD133+OCSLCs also showed increased sphereformation. Moreover, CD133+OCSLCs transfected with IL-17showed greater tumorigenesiscapacity in nude mice.3．NF-κB and p38MAPK signaling pathways are involved in IL-17-enhancedself-renewal of ovarian CD133+OCSLCs.On the basis of microarray analysis which used to compare the differential geneexpression profiles between IL-17-and PBS-treated A2780-derived CD133+OCSLCs，first,we found the NF-κB和MAPK signaling pathway by Signaling Net Array. Next, we screened8differentially expressed self-renewal related genes. QPCR further validated the mRNAchanges of the8self-renewal related genes. Most of these genes except TCL1A wereidentified as NF-κB and/or AP1target genes using the online Chip Transcription FactorSearch. This result is consistent with the finding by Signaling Net Array. Therefore, NF-κBand p38MAPK signaling pathways may be the mechanism of IL-17-enhanced self-renewalon ovarian CD133+OCSLCs.In order to further clarify that NF-κB and p38MAPK signaling pathways mediateIL-17-enhanced self-renewal on ovarian CD133+OCSLCs，further study was conducted.Western-blot, ELISA and immunofluorescence assay showed that IL-17stimulation wasassociated with a significant increase in the level of p65in the nuclear extracts at5min, while the level of p65in the cytoplasm decreased. To further verify the role of NF-κB signaling inIL-17-enhanced self-renewal, we used NF-κB inhibitor PDTC and/or P38inhibitor SB203580to block its activation during the treatment of IL-17. As expected, we found that the increasednumber of spheres caused by IL-17treatment could be abolished by PDTC and/or SB203580treatment. Therefore, in the self-renewal of CD133+OCSLCs, there forms a new mechanismof IL-17CD133+OCSLCs self-renewal promotion through activation of NF-κB and P38MAPK signaling pathways.ConclusionIL-17-producing cells (CD4+T cells and CD68+cells) were components of CD133+OCSLCs niche in ovarian cancer. IL-17could contact with IL17R on CD133+OCSLCs andpromote self-renewal in vitro and enhance tumorigenic potential of ovarian CD133+OCSLCsin xenograft mice. And these effects were mediated by NF-κB and p38MAPK signaling.