The Effects And Mechanisms Of NTS/NTR1 Signaling On Self-renewal And Invasion Of Glioblastoma Stem Cells

Background:Glioblastoma(GBM) is the most common primary malignant tumor in adult brain. Despite the improvement of neurosurgical skill and the implementation of comprehensive treatment strategy such as radiation, chemotherapy, biotherapy, the prognosis is not obviously improved for patients with glioblastoma. The primary challenge in clinical management of glioblastoma lies on the almost unavoidable tumor recurrence, because tumor cells could not be radically eliminated by surgical resection resulting from the invasive growth of tumors into the surrounding normal brain tissues. Glioma stem cells(GSCs) is a small fraction of tumor cells capable of self-renewal, multilineage differentiation and high tumorigenicity and is considered as the seed cells and root of glioblastoma initiation, progression, resistance to therapy and recurrence. Therefore, to explore the malignant biological characteristics and underlying mechanisms of GSCs sheds profound significance to find more effective and novel targets for glioblastoma treatment.Neurotensin(NTS) is a short neuropeptide composed of 13 amino acids, extensively expressed in central nervous system and digestive system. The biological functions of NTS are various such as modulation of inflammatory response and pain and NTS also play important roles in initiation and progression of a variety of diseases, which is mainly mediated by its high affinity receptor NTR1. In recent years, the intimate relationship between the aberrant change of NTS/NTR1 signaling and human malignant tumors such as lung cancer, colonic cancer and prostate cancer is established. Treatment of the specific antagonist of NTR1, SR48692, inhibits the proliferation, migration and invasion and promotes the therapy-sensitivity of malignant tumor cells. Previously,we found that NTS and NTR1 are highly expressed in gliomas and positively correlated with WHO grade and negatively correlated with prognosis of patients suffering from glioma. We further found that NTS/NTR1 signaling have promoting-effects of proliferation and invasion of glioma cells. Besides, compared to adhesion cultured glioma cells, an obvious increase of NTS was observed in GSCs and enhanced the tropism of microglia to GSCs. These results indicate that NTS/NTR1 signaling may exert important functions in regulation of malignant behaviors of GSCs. However, many questions are existed such as what are the precise roles and mechanisms of NTS/NTR1 in regulation of GSCs. So far, there are no reports to resolve these questions and it becomes urgent need to be further explored.Objective:1. to investigate the effects and mechanisms of NTS/NTR1 signaling on the self-renewal and invasive ability of GSCs.2. to observe the expression of NTS in glioblastoma tissues and analyze the correlation between NTS expression and clinicopathological features in glioblastoma and the value of NTS/NTR1 signaling in diagnosis and prognosis of patients with glioblastoma.Materials and Methods:1. GSCs from glioblastoma cell lines U87 and surgical resection tissues of glioblastoma(designated as GP-1 and GP-2) were obtained by sphere formation in serum-free medium. GSCs were identified by sphere-forming assay, stem cell molecular maker(Nestin and CD133) detection and tumorigenesis in BALB/c nude mice.2. Transwell experiment was used to detect the ability of invasion of GSCs and sphere-forming assay and soft agar assay were used to investigate the self-renewal ability of GSCs under varyng conditions(NTS treatment, SR48692 treatment and lentiviral-mediated NTR1 knockdown). Sphere-forming assay and limiting dilution assay were used to detect the self-renewal ability of GSCs. Flow cytometric analysis and western-blot were used to detect the expressions of stem cell markers, further confirming the regulatory role of NTS/NTR1 in self-renewal of GSCs. Subcutaneous model and Orthotopic xenografts of human glioblastoma in nude mice were established to investigate the effects of NTS/NTR1 on GSCs in vivo.3. The difference of chemokines and their receptors expressions were screened by quantitative PCR before and after NTR1 knockdown, and then verified by ELISA.4. After exogenous IL-8 treatment, knockdown of IL-8 by si RNA or overexpression of IL-8 in GSCs with NTR1 knockdown, transwell experiment,sphere-forming assay,soft agar assay, stem cell markers detection and subcutaneous mice model were carried out to investigate the role of IL-8 in NTS/NTR1-promoted self-renewal and invasion ability of GSCs.5. Using WB detects the activation of downstream signaling pathway(Akt,Erk1/2,Stat3) of NTS/IL-8 signaling. Transwell experiment,sphere-forming assay, and stem cell markers detection were carried out to investigate the role of CXCR1 in regulation of self-renewal and invasion ability of GSCs.6. Immunochemistry staining was performed to observe the expression pattern of NTS, IL-8, MMP-2, MMP-9 and Hif1α in glioblastoma tissues. Using Fisher exact test to analyze the correlation of NTS and IL-8 and Kaplan-Meier survival analysis was used to reveal the clinical value of NTS in glioblastoma tissues. We further analyze the correlation of MMP-2 MMP-9, Hif1α and NTS expression glioblastoma tissues.Results:1. We obtained GSCs from glioblastoma cell line U87 and glioblastoma tissues defined by high expression of stem cell markers(CD133 and Nestin) and highly tumorigenic ability in subcutaneous mice model of human glioblastoma.2. We found that the expression of NTS and NTR1 is increased in GSCs compared to differentiated gliomas. After treatment with specific antagonist for NTR1(SR48692) and Lentiviral mediated NTR1 knockdown, the self-renewal and invasion ability of GSCs were decreased as demonstrated by Transwell experiments, sphere-forming assay, soft agar assay and limiting-dilution assay. Simultaneously, the results of flow cytometric analysis and western-blot showed that the stem cell markers(Nestin, Oct3/4, Nanog and Sox2) were significantly reduced. Conversely, treated GSCs with NTS increased the self-renewal and invasion ability of GSCs. We further showed that NTR1 knockdown decreased the tumorigenic ability of GSCs in Subcutaneous model and Orthotopic xenografts of human glioblastoma in nude mice.3. RT-PCR results showed that NTR1 knockdown decreased the expression of IL-8 in GSCs to various extent but statistically significant. ELISA results found that NTR1 downregulation significantly reduced the secretion of IL-8 protein in U87 and GP-1 GSCs. Further, we found that the regulation of IL-8 by NTS is a relative common phenomenon in GSCs and is closely correlated with EGFR signaling.4. Downregulation of IL-8 by si RNA inhibited NTS-induced self-renewal and invasion of GSCs. Furthermore, either exgenous IL-8 treatemnt or over-expression of IL-8 in GSCs with NTR1 knockdown recovered the sphere-forming, colony-forming and invasion ability of GSCs in vitro and the tumorigenic ability in vivo.5. CXCR1/Stat3 participated in the promoting-effects of self-renewal and invasion ability of GSCs by NTS/IL-8 pathway. Treated GSCs either NTS or IL-8 increased the phosphorylation of Stat3 and there is no obvious alteration in Akt and Erk1/2. By contrast, knockdown of NTR1 decreased the phosphorylation of Stat3. In addition, treated with IL-8 to GSCs with NTR1 knockdown recovered the level of Stat3 phosphorylation. Both CXCR1 and CXCR2 were expressed in GSCs and treated GSCs with neutralization antibody for CXCR1 and CXCR2 significantly decreased the sphere-forming and invasive ability. Downregulation of CXCR1 by si RNA impaired sphere-forming and invasive ability and decreased stem cell markers of GSCs。6. The results from immunochemistry staining showed that NTS, IL-8 and MMP-9 were mainly expressed in cytoplasm and Hif1 alpha was located in nucleus. NTS expression positively correlated with expression of IL-8, MMP-9 and Hif1α. Although there was no obvious correlation betwwen MMP-2 and NTS expression, we found that the increase of MMP-2 in nucleus with the increase of WHO grade. WB results found that expressions of stem cell markers(Sox2, Oct3/4, Nanog) in glioblastoma tissues with NTS high expression were higher than glioblastoma tissues with NTS low expression. Further, Kaplan-meier survival analysis found that glioblastoma patients with NTS high expression showed shorter overall and progression-free survival time that that with NTS low expression.Conclusions:1. We found that high expression of NTS and NTR1 promotes self-renewal and invasion of GSCs isolated from glioblastoma cell line and primary glioblastoma cells. Most importantly, our results clarified the important roles of NTS/NTR1 signaling in regulation of GSCs for the first time.2. We found that NTS/NTR1 signaling is involved in regulation of IL-8 expression, and its mechanism is closely related to EGFR activation.3. We found that IL-8/CXCR1/Stat3 is an important downstream signaling participating in regulation of GSCs self-renewal and invasion ability by NTS/NTR1 signaling.4. We found that NTS exprssion positively correalated with IL-8 expression in glioblastoma and NTS high expression predicted a bad prognosis of patients with glioblastoma. Therefore, our results indicated that detecting the expression of NTS may function as a important molecular marker of prognosis for glioblastoma patients.