Minhui Xu

Glioma-initiating cells: A predominant role in microglia/macrophages tropism to glioma

The relationship between cancer-initiating cells and cancer-related inflammation is unclear. Exploring the interaction between glioma-initiating cells (GICs) and tumor-associated microglia/macrophages (TAM/Ms) may offer us an opportunity to further understand the inflammatory response in glioma and the cellular/molecular features of the GIC niche. Here，we reported a positive correlation between the infiltration of TAM/Ms and the density of GICs. The capacity of GICs to recruit TAM/Ms was stronger than that of adhesive glioma cells (AGCs) in vitro. In vivo experiments suggested that implantations formed by GICs had a higher level of TAM/M infiltration than those formed by AGCs. Our studies indicate a predominant role of GICs in microglia/macrophages tropism to glioma and a close positive correlation between the distribution of GICs and TAM/Ms. As an important part of cancer-related inflammation, TAM/Ms may participate in the architecture of the GIC niche.

Neurotensin signaling regulates stem-like traits of glioblastoma stem cells through activation of IL-8/CXCR1/STAT3 pathway

We recently found that neurotensin (NTS) and its primary receptor NTSR1 play a crucial role in glioblastoma cell proliferation and invasion. However, very little is known regarding the functional role of NTS/NTSR1 signaling in glioblastoma stem cells (GSCs). Here, we showed that NTSR1 is highly expressed in GSCs than its non-GSC counterparts. Pharmacological blockade with SR48692 or lentivirus mediated knockdown of NTSR1 efficiently reduced the sphere-forming ability and expression of stem cell markers such as nestin and Sox2 in GSCs isolated from glioblastoma cell line and glioblastoma tissues. Conversely, treated GSCs with NTS led to increase of tumor sphere formation. Mechanistically, we demonstrated that EGFR-dependent enhancement of IL-8 secretion is responsible for the effect of NTS signaling in the regulation of stem-like traits. Finally, we showed that NTSR1 or IL-8 knockdown decreased the phosphorylation of transcriptional factor STAT3 at Tyr705, which is a major transcription factor implicated in the regulation of GSC stem-like traits. Although both CXCR1 and CXCR2 inhibition reduced the tumor sphere formation, we found that CXCR1, but not CXCR2, is primarily responsible for STAT3 phosphorylation. Taken together, our findings suggest that NTS/IL-8/CXCR1/STAT3 signaling is crucial for the maintenance of stem-like traits in GSCs and provides a potential therapeutic target for glioblastoma therapy.

Sonic Hedgehog pathway is essential for neuroblastoma cell proliferation and tumor growth

Accumulated evidence suggests a major role for the activation of the Sonic Hedgehog (SHH) signaling pathway in the development of neural crest stem cells that give rise to the sympathetic nervous system. We therefore investigated the involvement of SHH signaling in the pathogenesis of neuroblastoma (NB), a common childhood malignant tumor of the sympathetic nervous system. Inhibition of SHH signaling by cyclopamine induced apoptosis and blocked proliferation in all major types of NB cells, and abrogated the tumorigenicity of NB cells. Our study has revealed a molecular mechanism for the persistent activation of the SHH pathway which promotes the development of NB, and suggests a new approach for the treatment of this childhood malignant tumor.

Neurotensin signaling stimulates glioblastoma cell proliferation by upregulating c-Myc and inhibiting miR-29b-1 and miR-129-3p

BACKGROUND Neurotensin (NTS) and its primary receptor NTSR1 are implicated in cancer progression. Aberrant expression of NTS/NTSR1 contributes to the proliferation of glioblastoma cells; however, the mechanism is not fully understood. METHODS Microarray and real-time PCR were performed to identify the NTS-regulated micro (mi)RNAs. The targets of the miRNAs were identified by luciferase assays and immunoblot analysis. The c-Myc binding sites in the miR-29b-1 and cyclin-dependent kinase (CDK)4 promoters were identified through chromatin immunoprecipitation assay. Cell proliferation was evaluated by Cell Counting Kit-8 assay and flow cytometry analysis. An orthotopic xenograft model demonstrated the role of NTS/NTSR1 and miRNAs in glioblastoma growth in vivo. RESULTS Pharmacological inhibition or small interfering NTSR1 treatment blocked glioblastoma cell cycle progression in the G1 phase with a concomitantly decreased expression of CDK6, CDK4, and c-Myc. Knockdown of NTSR1 increased the expression of miR-29b-1 and miR-129-3p, which were responsible for the decreased CDK6 expression. NTS/NTSR1 signaling activated the transcription factor c-Myc in U87 cells, leading to increased CDK4 expression and repressed miR-29b-1 expression. Knockdown of NTSR1 decreased the glioblastoma growth in vivo and significantly prolonged the survival time of the tumor-bearing mice, an effect that can be largely reversed by antagomir. CONCLUSIONS Our study showed a novel regulatory mechanism of NTS/NTSR1, an upstream signaling of miRNAs and c-Myc, in glioblastoma progression. The inhibition of the NTSR1 function or the upregulation of miR-29b-1 and miR-129-3p expression impaired glioma cell proliferation. These results suggested that the NTS/NTSR1/c-Myc/miRNA axis may be a potential therapeutic target for glioblastoma therapy.

MicroRNAs and cell cycle of malignant glioma

The control of malignant glioma cell cycle by microRNAs (miRNAs) is well established. The deregulation of miRNAs in glioma may contribute to tumor proliferation by directly targeting the critical cell-cycle regulators. Tumor suppressive miRNAs inhibit cell cycle through repressing the expression of positive cell-cycle regulators. However, oncogenic miRNAs promote the cell-cycle progression by targeting cell-cycle negative regulators. Recent studies have identified that transcription factors had involved in the expression of miRNAs. Transcription factors and miRNAs are implicated in regulatory network of glioma cell cycle, the deregulation of these transcription factors might be a cause of the deregulation of miRNAs. Abnormal versions of miRNAs have been implicated in the cell cycle of glioma. Based on those, miRNAs are excellent biomarker candidates and potential targets for therapeutic intervention in glioma.

Neurotensin promotes the progression of malignant glioma through NTSR1 and impacts the prognosis of glioma patients

BackgroundThe poor prognosis and minimally successful treatments of malignant glioma indicate a challenge to identify new therapeutic targets which impact glioma progression. Neurotensin (NTS) and its high affinity receptor (NTSR1) overexpression induces neoplastic growth and predicts the poor prognosis in various malignancies. Whether NTS can promote the glioma progression and its prognostic significance for glioma patients remains unclear.MethodsNTS precursor (ProNTS), NTS and NTSR1 expression levels in glioma were detected by immunobloting Elisa and immunohistochemistry assay. The prognostic analysis was conducted from internet by R2 microarray platform. Glioma cell proliferation was evaluated by CCK8 and BrdU incorporation assay. Wound healing model and Matrigel transwell assay were utilized to test cellular migration and invasion. The orthotopic glioma implantations were established to analyze the role of NTS and NTSR1 in glioma progression in vivo.ResultsPositive correlations were shown between the expression levels of NTS and NTSR1 with the pathological grade of gliomas. The high expression levels of NTS and NTSR1 indicate a worse prognosis in glioma patients. The proliferation and invasiveness of glioma cells could be enhanced by NTS stimulation and impaired by the inhibition of NTSR1. NTS stimulated Erk1/2 phosphorylation in glioma cells, which could be reversed by SR48692 or NTSR1-siRNA. In vivo experiments showed that SR48692 significantly prolonged the survival length of glioma-bearing mice and inhibited glioma cell invasiveness.ConclusionNTS promotes the proliferation and invasion of glioma via the activation of NTSR1. High expression levels of NTS and NTSR1 predict a poor prognosis in glioma patients.

Glioma-derived T Cell Immunoglobulin- and Mucin Domain-containing Molecule-4 (TIM4) Contributes to Tumor Tolerance

Background: We investigated the role of TIM4 in the induction of regulatory T cells (Tregs) in tumors. Results: High TIM4 levels were detected in glioma tissue and contributed to tumor-specific Treg development. Conclusion: TIM4 is important in inducing Tregs in gliomas. Significance: TIM4 may be a target in glioma treatment. Tumor tolerance plays a critical role in tumor growth and escape from immune surveillance. The mechanism of tumor tolerance development is not fully understood. Regulatory T cells (Tregs) play a critical role in tumor tolerance. TIM4 (T cell immunoglobulin- and mucin domain-containing molecule-4) is involved in immune regulation. We investigated the role of TIM4 in the induction of Tregs in tumors. Surgically removed glioma tissue and peripheral blood samples were obtained from 25 glioma patients. Immune cells were isolated from the tissue and blood samples. Confocal microscopy was employed to detect macrophages phagocytosing apoptotic T cells. The generation of tumor-specific Tregs and the immune suppression function of Tregs were observed in cell culture models. High levels of TIM4 were detected in glioma-derived macrophages. Phosphatidylserine (PS) was detected in glioma-derived T cells; naïve T cells expressed low levels of PS that could be up-regulated by hypoxia. Glioma-derived macrophages phagocytosed PS-expressing T cells, gaining the tolerogenic properties, which could induce tumor-specific Tregs; the latter could suppress tumor-specific CD8+ T cells. We conclude that macrophage-derived TIM4 plays an important role in the induction of Tregs in gliomas, which may play an important role in tumor tolerance.

Implantation of GL261 neurospheres into C57/BL6 mice: A more reliable syngeneic graft model for research on glioma-initiating cells

Recent studies have demonstrated that inflammatory cells and inflammatory mediators are indispensable components of the tumor-initiating cell (TIC) niche and regulate the malignant behavior of TICs. However, conventional animal models for glioma-initiating cell (GIC) studies are based on the implantation of GICs from human glioblastoma (GBM) into immunodeficient mice without the regulation of immune system. Whether animal models can mimic the cellular microenvironment of malignancy and evaluate the biological features of GICs accurately is unclear. Here, we detected the biological features of neurosphere-like tumor cells derived from the murine GBM cell line GL261 (GL261-NS) and from primary human GBM (PGBM-NS) in vitro, injected GL261-NS into syngeneic C57/BL6 mouse brain and injected PGBM-NS into NOD/SCID mouse brain, respectively. The tumorigenic characteristics of the two different orthotopic transplantation models were analyzed and the histological discrepancy between grafts and human primary GBM was compared. We found that GICs enriched in GL261-NS, GL261-NS and PGBM-NS exhibited increased GIC potential and enhanced chemoresistance in vitro. GL261-NS was significantly more aggressive compared to GL261 adhesive cells (GL261-AC) in vivo and the enhanced aggression was more significant in syngeneic mice compared to immunodeficient mice. The discrepancy of tumorigenicity between GL261-NS and GL261-AC in C57/BL6 mice was also larger compared to that between PGBM-NS and PGBM-AC in immunodeficient mice. Syngrafts derived from GL261-NS in C57/BL6 mice corresponded to the human GBM histologically better, compared with xenografts derived from PGBM-NS in NOD/SCID mice, which lack inflammatory cells and inflammatory mediators. We conclude that the inflammatory niche is involved in the tumorigenicity of GICs and implantation of GL261-NS into C57/BL6 mice is a more reliable syngeneic graft model for in vivo study on GICs relative to the immunodeficiency model.

Oncogenic role of Neurotensin and Neurotensin receptors in various cancers

Neurotensin (NTS) has long been recognized as a neurotransmitter or neuromodulator in the central nervous system and as an endocrine agent in the periphery via actions mediated through neurotensin receptors (NTSRs). Many studies support a role for NTS in the endocrine, autocrine and paracrine growth stimulation of cancer, with oncogenic actions described for NTS in different types of cancers and cancer cell lines at each step of cancer progression, ranging from tumour growth and survival to metastatic spread. The mechanisms underlying the effects of the NTS/NTSR system in cell proliferation, migration and invasion, as well as the anti‐apoptotic effects of this system, have been elucidated in different types of cancers, and include mitogen‐activated protein kinases, phosphatidylinositol 3‐kinase and RhoGTPases. The present mini review summarizes recent findings relating to the oncogenic function of the NTS/NTSR system.

Interference with NTSR1 Expression Exerts an Anti-Invasion Effect via the Jun/miR-494/SOCS6 Axis of Glioblastoma Cells

Background/Aims: Glioblastoma is the most common and aggressive brain tumor and carries a poor prognosis. Previously, we found that neurotensin receptor 1 (NTSR1) contributes to glioma progression, but the underlying mechanisms of NTSR1 in glioblastoma invasion remain to be clarified. The aim of this study was to investigate the molecular mechanisms of NTSR1 in glioblastoma invasion. Methods: Cell migration and invasion were evaluated using wound-healing and transwell assays. Cell proliferation was detected using CCK-8. The expression of NTSR1, Jun, and suppressor of cytokine signaling 6 (SOCS6) was detected using western blotting. The expression of miR-494 was detected by Quantitative real-time PCR. Chromatin immunoprecipitation assay was performed to examine the interaction between Jun and miR-494 promoter. Dual-luciferase reporter assay and western blotting were performed to identify the direct regulation of SOCS6 by miR-494. An orthotopic xenograft mouse model was conducted to assess tumor growth and invasion. Results: NTSR1 knockdown attenuated the invasion of glioblastoma cells. Jun was positively regulated by NTSR1, which promoted miR-494 expression through binding to miR-494 promoter. SOCS6 was confirmed as a direct target of miR-494, thus, NTSR1-induced miR-494 upregulation resulted in SOCS6 downregulation. Both miR-494 and SOCS6 were involved in the NTSR1-induced invasion of glioblastoma cells. In vivo, tumor invasion and growth were inhibited by NTSR1 knockdown, but were restored with miR-494 overexpression. Conclusion: NTSR1 knockdown inhibited glioblastoma invasion via the Jun/miR-494/SOCS6 axis.