- Open Access
Neurotensin promotes the progression of malignant glioma through NTSR1 and impacts the prognosis of glioma patients
- Qing Ouyang†1, 2,
- Xueyang Gong†2,
- Hualiang Xiao†4,
- Ji Zhou1,
- Minhui Xu1,
- Yun Dai5,
- Lunshan Xu1,
- Hua Feng3,
- Hongjuan Cui2Email author and
- Liang Yi1Email author
© Ouyang et al.; licensee BioMed Central. 2015
- Received: 16 August 2014
- Accepted: 5 January 2015
- Published: 3 February 2015
The 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.
NTS 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.
Positive 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.
NTS 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.
For the last decade, the improvement of neurosurgery, radiotherapy and chemotherapy have prolonged the survival time of malignant glioma patients. However, the high recurrence rate still results in the high death rate of patients . Vigorous proliferation and extensive invasion make it extremely difficult to completely clear out glioma. The high cellular invasiveness and the residual glioma cells become the sources of recurrence . Inhibition of cellular proliferation and invasiveness has always been a basic strategy to combat malignance. However, the effective therapies that suppress the growth and invasiveness of gliomas are limited, and the underlying mechanisms need to be investigated further.
Neurotenin (NTS) is present in the central nervous system (CNS) and in periphery. High expression level of NTS can be detected in hypothalamus, median eminence, pituitary stalk, substantia nigra, locus coeruleus, raphe nuclei and brainstem structure, especially in amygdale, arcuate nucleus and limbic system which are closely related to psychological activity. However, NTS has the low expression level in cerebral cortex,hippocampus, basal ganglion and thalamus [3,4]. It acts as a neurotransmitter function to inhibit dopaminergic pathways and induce a serial of neurological effects. In the periphery, NTS is mainly secreted by endocrine N-cells of the gastrointestinal tract and plays the role of a neurocrine hormone to regulate the postprandial digestive process. It inhibits gut motility and gastric acid secretions, stimulates the pancreatic and biliary secretions and improves the fatty acid ingestion [5,6].
It has reported that NTS and its high-affinity neurotensin receptor 1 (NTSR1) overexpress in several types of cancer and malignant cell lines. Accumulating evidences also confirm that the activation of NTS/NTSR1 complex results in cancer progression and poor prognosis in breast cancer, malignant pleural mesothelioma, and head and neck squamous cell carcinomas [6,7]. NTSR1 activates at least three major pathways in cancer, which are small GTPases activation inducing cellular mobility, intracellular Ca2+ mobilization involving in gene regulation and proto-oncogene serine/threonine-protein kinase/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf-1/Mek/Erk) cascade inducing cell proliferation . Our previous research found that NTS is highly upregulated in glioma stem cells and promotes the motility of microglia . However, as a neuropeptide in the CNS, the potential biological functions of NTS/NTSR1 and their downstream signaling pathway in glioma are unclear.
Here, we detected the expression levels of NTS and NTSR1 in glioma specimens and investigated the relationship between the expression levels and the patients’ prognosis. The role of the NTS/NTSR1/Erk1/2 signal axis in the proliferation and invasiveness of malignant glioma cells was tested in vitro. We established intracranial orthotopic transplantation gliomas in mice, and analyzed the impact of the NTSR1 specific inhibitor SR48692 on the biological behaviors of the glioma cells and the survival time of the glioma-bearing mice. Our results highlighted that NTS promotes the proliferation and invasiveness of malignant glioma cells through NTSR1 and its downstream signaling molecules, leading to Erk1/2 phosphorylation. We firstly reported that high levels of NTS or NTSR1 expression were correlated with a poor prognosis in the glioma patient, which would be a potential target for glioma treatment and need to be further investigated.
NTS and NTSR1 expression patterns in human glioma specimens
High NTS and NTSR1 expression indicated a poor prognosis for glioma patients
The Sun dataset  from R2 microarray platform includes 153 glioma cases with different histological grades (grade II, III, and IV). The microarray data have been submitted to the Gene Expression Omnibus (GEO) public database at NCBI (GSE4290). The result of Affymetrix HU133 Plus 2.0 confirmed that increased NTS expression significantly was correlated with advanced tumor stages in the Sun dataset (Figure 2B).
Since NTS and NTSR1 expression was associated with the pathological grade of glioma, we investigated the possibility of NTS or NTSR1 as a prognostic marker for glioma patients. We found that high NTS and NTSR1 mRNA expression indicated a poor outcome in the Gravedeel dataset , which includes a cohort of 273 glioma patients (Figure 2C). Kaplan–Meier analysis of the overall survival for this dataset showed that the 3-year survival rates for patients with high expression (167 cases) and low expression (106 cases) of NTS mRNA was 25% and 39% (p = 0.018), respectively, and the 5-year survival rates for these patients was 17% and 28% (p = 0.013), respectively. Meanwhile, the 3-year survival rates for patients with high expression (132 cases) and low expression (141 cases) of NTSR1 mRNA was 18% and 36% (p = 0.011), respectively, and the 5-year survival rates for these patients was 15% and 25% (p = 0.022), respectively. We confirmed that high NTS and NTSR1 expression were both associated with poor prognosis, whereas low NTS and NTSR1 expression were associated with good outcome (Figure 2C). The prognostic value of NTS was also verified in Rembrandt database, especially in “Astrocytoma” sub-database. However, NTS had no relationship with the overall survival probabilities of de novo GBM patients in TCGA database, but had a significantly negative relationship with their progression-free survival probability (Additional file 4: Figure S2).
NTS promoted malignant glioma cell proliferation and invasion through NTSR1
NTSR1 activated the phosphorylation of Erk1/2 to promote the proliferation and invasiveness of glioma cells
We found that Raf-1/Mek/Erk1/2 pathway was activated after NTS stimulation, but not other molecules and pathways, including p15, p16, p38, pAKT, mTOR, pSmad2/3, pJAK2 and pSTAT3 (Additional file 6: Figure S4). The phosphorylation of Erk1/2 could be inhibited by the NTS neutralizing antibody, the NTSR1-selective antagonist SR48692 and NTSR1-siRNA; these results suggested that the elevated phosphorylation of Erk1/2 stimulated by NTS was induced in an NTSR1-dependent manner (Figure 5C, D). Additionally, the MEK1/2-selective inhibitor U0126 reduced the amount of BrdU-positive cells and invasive cells induced by NTS (Figure 5E, F). These results indicated that the above effects of NTS and NTSR1 in glioma cells were induced by the activation of the MEK/ERK signaling pathway.
NTSR1-selective antagonist SR48692 inhibited glioma progression in vivo
We also assessed MRI detection to monitor the growth of orthotopic xenografts. Tumor dimensions were determined from the MR image and Tv was calculated. We found that SR48692 could significantly inhibit the tumor growth in vivo (Figure 6B). Kaplan-Meier survival analysis confirmed that treatment with SR48692 significantly prolonged the survival time of the C57/BL6 mice bearing syngeneic GL261 gliomas (median survival of 18.6 ± 3.6 days for DMSO versus 33.0 ± 5.4 days for 10 mg/kg SR48692 treatment) (Figure 6B). Proton magnetic resonance spectroscopy (MRS) can analyze the chemical component in specific tissue region noninvasively. The areas of choline (Cho) peak, n-acetylaspartic acid (NAA) peak and creatine (Cr) peak represent the concentrations of these substances, which reflect the metabolic status of cell and organization. The higher Cho/NAA ratio and Cho/Cr ratio, the more proliferative and malignant in the detected region. In order to evaluate the invasion of xenografts, we carried out MRS detection at the borderline of tumors and found that SR48692 treatment caused a drop in the MRS-detectable Cho/NAA ratio and Cho/Cr ratio in peritumoral tissues (Additional file 7: Figure S5). This meant that there were less malignant cells invading into the preitumoral tissue of SR48692 treatment group (Figure 6B).
Meanwhile, the pathological observation as also performed. In the control group, the margins between the tumor and the normal brain tissue were rough and unclear, indicating an intraparenchymal invasion pattern of glioma. In contrast, the margins of the tumors in the 5 mg/kg SR48692 treatment group were smooth and did not show invasive characteristics of malignant glioma, which were in accord with the MRS detection (Figure 6C). Additionally, we tested the expression of NTS in the xenografts using IHC. NTS was highly expressed in the xenografts of the control group, whereas it was rarely expressed in glioma following SR48692 treatment (Figure 6D). These results indicated that the inhibition of NTSR1 could impair glioma progression in vivo.
NTS can be detected in the central nervous system and in the periphery. It produces a wide range of physiological and pharmacological effects . NTS regulates the release of luteinizing hormone and prolactin and has significant interaction with the dopaminergic system, which induces a variety of effects, including: analgesia, hypothermia and increased locomotor activity. In the periphery, neurotensin is found in endocrine cells of the small intestine, where it leads to secretion and smooth muscle contraction. Meanwhile, it also act as a paracrine and endocrine modulator of the cardiovascular system [12,13]. So far, three types of NTS receptors have been discovered, including two G protein coupled receptors (NTSR1 and NTSR2) and a non-specific sorting receptor (NTSR3/sortilin) [13,14]. Recently, it has been shown that NTS is very important in the oncogenic progression of several types of cancer cells [6,7]. NTS has been shown to have growth stimulatory and pro-invasive effects in breast cancer [15,16] and malignant tumors in the digestive system, including hepatomas , colon cancer [18,19] and pancreatic cancer [20,21]. Most of its functions in malignant progression are mediated by NTSR1, its high affinity receptor . However, the role of NTS and NTSR1 in malignant glioma has only rarely been reported. Our results confirmed the pro-growth and pro-invasion roles of NTS in glioma progression. NTS and NTSR1 are highly expressed in glioma tissue, especially in glioblastomas, and their expression levels are positively correlated with the pathological grade of the gliomas. We first reported that high levels of NTS and NTSR1 expression predict a decreased survival rate in glioma patients. The proliferation and invasiveness of malignant glioma cells could be suppressed by inhibiting the interaction between NTS and NTSR1 in vitro. A similar phenomenon was also observed in the glioma-bearing mice model. SR48692, a NTSR1 specific antagonist, inhibited the invasiveness of orthotopically implanted glioma cells in the mouse brain and prolonged the survival time of the experimental animals. We reported that NTS was expressed at higher levels in glioma stem cells (GSCs) than in differentiated glioma cells . It has been confirmed that GSCs exhibit self-renewal capacity and a high invasive potential, which results in the recurrence of glioma . Considering that NTS can induce neoplastic progression, the role of NTS and NTSR1 in the malignant biological behaviors of GSCs should be investigated in future studies.
In recent decades, it has been shown that inflammation plays an important role in tumorigenesis and tumor progression [23,24]. Intensive infiltration of immune cells and high levels of inflammatory mediators are often found in tumor sites and contribute to malignant biological behaviors. Tumor-associated-microglia/macrophages (TAM/Ms) are the largest population of infiltrating inflammatory cells in glioma . We have reported that NTS played a predominate role in TAM/M recruitment in glioma . It has been confirmed that microglia do not express NTSR1 and NTSR2 but NTSR3, which results in the migration of microglia in a PI3K/MAPK-dependent mechanism . Once recruited to the glioma microenvironment, TAM/Ms promote the proliferation of glioma cells in the stromal areas, enhance the invasiveness of glioma cells at the tumor margins and stimulate angiogenesis in the perivascular areas . Additionally, NTS enhances the release of pro-tumoral inflammation mediators, especially interleukin-8, from tumor cells and inflammatory cells. The enhanced release of IL-8 induced by NTS/NTSR1 has been confirmed in pancreatic cancer , hepatocellular carcinomas [17,29] and colon cancer [30,31]. IL-8 widely contributes to the angiogenesis and invasion of tumor cells . Thus, the mechanism of NTS-induced glioma progression is complex and may be divided into three parts. First, NTS can promote the proliferation and invasiveness of glioma cells directly through NTSR1. Second, NTS enhances the release of inflammatory mediators to contribute to glioma progression. Third, it promotes the growth, invasion, angiogenesis and immune evasion of glioma by inducing TAM/M infiltration indirectly through NTSR3.
NTSR1, a seven-transmembrane domain G-protein-coupled receptor, has been shown to bind to G subunits that activate phospholipase C (PLC) . Then, PLC can induce the production of inositol triphosphate (IP3) and stimulate Protein Kinase C (PKC). In various types of tumor cells, the two above-mentioned pathways, especially PKC downstream signaling, regulate the effects of NTS in tumor cells . However, the signaling pathway inducing the malignant properties of NTS in glioma is unclear. Previous researches also confirmed that the effect of NTS/NTSR1 stimulation on cell growth mainly mediated by MEK/ERK1/2 phosphorylation pathway in colon cancer  and pancreatic cancer . We found that the MEK1/2-selective inhibitor U0126 could inhibit the proliferation and invasiveness induced by NTS. NTS stimulated Erk1/2 phosphorylation, and the elevated phosphorylation level was reversed by NTS-NA and SR48692. These results indicated that the oncogenic effects of NTS on glioma involved the activation of the MAPK signal pathway. In NTS-stimulated pancreatic cancer cells, two pathways could induce MAPK cascade activation in a PKC-dependent manner [34,35]. PKC could directly stimulate Raf-1, which upregulates MEK/ERK phosphorylation . Meanwhile, PKC could also induce protein kinase D1 (PKD1) activity, which can activate the phosphorylation of Erk1/2 and NF-ΚB . The signaling effector that mediates the activation of the MAPK cascade in NTS-stimulated glioma cells should be investigated further. Additionally, epidermal growth factor receptor (EGFR) transactivation has been reported in NTS-stimulated prostatic cancer cell . Dupouy et al. reported the progression of breast cancer induced by NTS/NTSR1 in an experimental mice model ensues following EGFR, HER2, and HER3 over-expression and autocrine activation . Younes et al. also reported NTS autocrine and/or paracrine regulation causes EGFR, HER2, and HER3 over-expression and activation in lung tumor cells . Prolonged ERK phosphorylation has also been detected in pancreatic cancer cells due to the synergistic stimulation of NTS and EGF . There are at least two pathways through which NTSR1 can mediate the activation of EGFR downstream signaling. First, NTS can induce the release of EGF-like ligands to stimulate EGFR. Meanwhile, NTS can stimulate the phosphorylation of EGFR at Tyr845 by c-Src through a PKC-dependent pathway . High EGFR expression and mutations in EGFR are prevalent in malignant glioma. The amplification and mutation of EGFR has been detected in 40%-50% of GBMs and oligodendrogliomas , EGFRvIII, which is a constitutively active EGFR mutant, can be detected in 12%-16% of GBM by IHC. The activation of the EGFR signaling pathway is involved in most of the malignant biological behaviors of glioma. The amplification of EGFR and the expression of EGFRvIII are biomarkers of poor prognosis in glioma patients . It would be very valuable to know whether the cooperative relationship between NTSR1 and EGFR system exists in malignant glioma, and what its underlying molecular mechanism is. Because the NTS/NTSR1-induced transactivation of the EGFR signaling pathway may complicate EGFR-targeted therapies in malignant glioma.
Our study was approved by the Ethics Committee of Daping Hospital, Third Military Medical University, Chongqing, P.R. China. Thirty consecutive, surgically resected astrocytomas were identified from the surgical sample database of the Neurosurgery Department of Daping Hospital (Additional file 1: Table S1). None of the patients had undergone chemotherapy or radiotherapy prior to surgery, except two cases of recurrent glioblastoma. All tumor specimens were selected and classified based on the WHO Grade criteria. The peritumoral tissue and relatively normal brain tissue of GBM patient were acquired in fistulization procedure of tumorectomy.
Analyses of patient data
Gene expression datasets were obtained by R2 microarray analysis and the visualization platform (http://hgserver1.amc.nl/cgi-bin/r2/main.cgi) and Rembrandt database (https://caintegrator.nci.nih.gov/rembrandt/). Affymetrix HU133 Plus 2.0 microarrays were used, the Affymetrix probe-sets for NTS and NTSR1 were 206291_at and 207360_s_at respectively. Kaplan–Meier analysis was conducted online, and the resulting survival curves and P values (log-rank test) were downloaded from internet. All cutoff values for separating the high and low expression groups were determined using the online R2 microarray platform algorithm [9,10].
Cell culture, drug treatment and siRNA-transfection
The murine glioma cell line GL261 and the human glioma cell line U87 were obtained from the American Type Culture Collection (ATCC, USA) and cultured in DMEM/F12 (Hyclone) supplemented with 10% fetal bovine serum (FBS, Sigma), penicillin and streptomycin (Sigma). The cells were plated and incubated at 37°C to achieve 25–50% confluency. Silencer® Select Pre-Designed siRNA against NTSR1 (NTSR1-siRNA, siRNA ID: 156980 and 143658) and a control-siRNA (negative control #1 siRNA, catalog #: 4390843) were purchased from Ambion (Austin, TX, USA). GL261 cells and U87 cells were transfected with NTSR1-siRNA using Lipofectamine™ RNAiMAX according to the manufacturer’s protocol. After 24 hours, the glioma cells were prepared for related experiments. DMEM/F12 with L-glutamine was used for all serum starvation experiments. The cells were rinsed with phosphate-buffered saline (PBS) and replaced with serum-free medium for 24 hours. For proliferation and 5-bromo-2′-deoxyuridine (BrdU) incorporation experiments, exogenous NTS and/or inhibitors, including the NTS neutralizing antibody (NA-NTS) (2 ng/ml, N2177-01, Biomol, Germany) and SR48692, were added to the medium at the beginning of the serum starvation period. For western blot analysis, the cells were treated with exogenous NTS and/or inhibitors immediately before cell lysis.
Cell proliferation and DNA synthesis assays
GL261 cells (2×103 cells/well) and U87 cells (2×103 cells/well) were seeded in 96-well plates and serum-starved for 24 hours. Cell proliferation was evaluated using a CCK8 (Cell Counting kit-8) kit according to the manufacturer’s protocol. Briefly, 10 μl CCK8 solution (Dojindo, Kumamoto, Japan) was added to each well, and the samples were incubated at 37°C for 2 hours before the absorbance was measured at 450 nm wave length. Each experimental condition, including blank wells, control wells, and control wells treated with drugs, were assayed in duplicate, and all experiments were performed at least three times.
For DNA synthesis assays, the cells were serum-starved for 24 hours. BrdU assays were performed using BrdU kits (Sigma, St. Louis, MO) as indicated by the manufacturer. Briefly, after serum starvation, BrdU was dissolved in PBS at a final concentration of 1 mg/ml, and 5 μl was added into each well. For each time point, BrdU was mixed into the cells for at least 1 hours, and the cells were stained with primary antibody to BrdU and Cy3-conjugated secondary antibody. The cells were then counterstained with 4′ 6-diamidino-2-phenylindole (DAPI). Fluorescent images were captured using a fluorescence microscope (Carl Zeiss Axio Observer).
Wound healing and Transwell assays
For the wound healing assays, GL261 cells were plated in 6-well dishes. 24 hours after cells reached 100% confluence, 10 ug/ml mitomycin C was added for 2 hours to eliminate the effect of proliferation, and a scratch was made in the monolayer with a pipette tip. The cells were maintained in low-serum medium (0.1% FCS), and pictures were taken 0, 36 and 72 hours respectively.
Cell invasiveness was studied using a 24-well matrigel transwell chamber assay plate, with an 8 μm pore size membrane (BD Falcon, USA). Matrigel was prepared according to the manufacturer’s instructions. In brief, 10 ug/ml mitomycin C was added to pretreat GL261 cells (5 × 104 cells/100 μl) or U87 cells (5 × 104 cells/100 μl) in serum-free DMEM/F12 medium (Hyclone) for 2 hours. Then, cells were seeded into the upper well of the insert, the lower well was filled with 600 μl of the different conditioned media. After the chambers were incubated at 37°C in a 5%CO2 incubator for 20 hours. Invasiveness was calculated by the number of cells invaded through the matrigel chamber and adhered to the bottom of the filter which were stained with crystal violet. Nine fields at 100× magnification were counted for each well. Each experiment was performed in triplicate.
Elisa, Immunoblotting, immunofluorescence and immunohistochemistry
The NTS peptide levels in glioma were measured by the ELISA method (CUSABIO’ Human Neurotensin ELISA Kit), according to the manufacturer’s instructions. 100 mg tissue was rinsed with 1X PBS, homogenized in 1 ml of 1X PBS and stored overnight at −20°C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 × g, 2 - 8°C. The supernate was removed and assayed immediately. The limit of detection was 15.6 pg/ml-1000 pg/ml with specificity that recognizes both natural and recombinant human NTS and sensitivity of < 3.9 pg/ml. There were three duplicative holes for every sample in Elisa assay.
Immunoblotting was conducted according to the standard procedures outlined in the Additional file 8. The membrane was incubated with antibodies against ProNTS (1:500; N2177-10, Biomol, Germany) or NTSR1 (1:1000; ab117592, Abcam, USA) at 4°C for 12 h. Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit immunoglobulin G (1:500; A0216&A0208, Beyotime, China) were used as secondary antibodies. Proteins were visualized using a Super Signal West Pico chemiluminescence kit (Pierce) and were quantified using the Odyssey system and software (LI-COR Biosciences). For immunofluorescence, the cells were fixed with 4% paraformaldehyde (PFA). Two kinds of primary antibodies for NTSR1 (ab183088 & ab117592, Abcam,USA) were used at 1:300 dilution on GL261 cells and U87 cells. Cy3-labeled goat anti-mouse IgG (1:500; A0521, Beyotime, China) for GL261 and FITC-labeled goat anti-rabbit IgG (1:500; A0562, Beyotime, China) for U87 were used as secondary immunofluorescence antibodies. The nuclei were stained with DAPI. Fluorescent images were captured using a fluorescence microscope (Carl Zeiss Axio Observer).
Immunohistochemistry (IHC) was performed on paraffin-embedded sections. The tumor sections were incubated with primary antibodies for NTS (1:500, N2177-01, Biomol, Germany) and for NTSR1 (1:200, ab117592, Abcam, USA), followed by detection using a ChemMate Detection kit (Dako, Denmark). A positive reaction was indicated by brown color using DAB, and the cells were counterstained with hematoxylin.
Syngeneic orthotopic glioma implantation and Magnetic resonance imaging (MRI) experiments
All procedures involving mice were conducted in accordance with the Guidelines of Animal Experiments of Third Military Medical University. All mice were purchased from Experimental Animal Center of Third Military Medical University. GL261 cells (5 × 104) were injected orthotopically into the brains of 6-week old female C57BL/6 mice (n = 24). The detailed measurement of intracranial tumors was listed in Additional file 8. 3 days after injection, 20 mice were randomly divided into 4 groups of 5 animals each. The groups were treated through i.p. injection with 2 mg/kg, 5 mg/kg, 10 mg/kg SR48692 respectively. SR48692 was resuspended in DMSO which was used as control. The mice were treated every two days for a total of five times. The weigh curves in different groups were recorded and showed in Additional file 5: Figure S3E. The survival periods of the mice were recorded. The brains of the mice were collected, fixed in formalin, and paraffin-embedded. The rest 4 mice were also divided into two groups at 3 days after implantation and treated with DMSO or SR48692 (10 mg/kg) respectively. 10 days after tumor cell injection, these 4 mice were euthanized and their brains were removed and processed for histopathologic analysis. The MRI equipment was a Bruker Biospec 7.0 Tesla imaging system (Bruker BioSpin MRI GmbH, Germany). MRI experiments were carried out under general anesthesia (1–2% isoflurane, 0.8–1.0 L/min O2). Mice were imaged at 7 days after the cells were injected and then every 3 days until 19 days after implantation. Tumor dimensions were determined from the MR image and tumor volume (Tv) was calculated using the formula: Tv = (π/6) × length × width × depth. MRS was used to monitor the glioma invasion. We placed a voxel (1 mm × 1 mm × 1 mm) at the borderline of tumors and repeated the MRS test for several times. Cho/NAA ratios and Cho/Cr ratios in these regions were calculated to assess the invasion of glioma cells in peritumoral tissues.
All statistical analyses were performed using the SPSS 13.0 statistical package (SPSS, Chicago, IL, USA). The statistical significance of the differences among more than three groups was determined using one way analysis of variance (ANOVA) with Bonferroni’s Multiple Comparison to compare each two groups. The statistical significance of the differences between two groups was determined using a t test. Survival data were analyzed using the log-rank test. Differences were considered significant when p < 0.05.
Expression levels of NTS/NTSR1 positively correlated with glioma pathological grade.
High expression levels of NTS/NTSR1 indicate a worse prognosis in glioma patients.
NTS/NTSR1 signaling regulates the proliferation and invasiveness of glioma cells.
NTSR1 antagonist, SR48692, prolongs the survival periods of glioma-bearing mice.
This study was supported by the National Natural Science Foundations of China (NSFC, Nos. 81270039 and 30901538), the opening research project from the State Key Laboratory of Silkworm Genome Biology (no. SKLSGB201200014), the National Natural Science Foundations from Chongqing Science and Technology Committee (no.cstc2012jjA0306) and the National Basic Research Program of China (no.2012CB114603).
National Natural Science Foundations of China (NSFC, Nos. 81270039 and 30901538), the opening research project from State Key Laboratory of Silkworm Genome Biology (no. SKLSGB201200014), the National Natural Science Foundations from Chongqing Science and Technology Committee (no.cstc2012jjA0306) and the National Basic Research Program of China (no.2012CB114603).
- Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, et al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol. 2009;10:459–66.View ArticlePubMedGoogle Scholar
- Reardon DA, Groves MD, Wen PY, Nabors L, Mikkelsen T, Rosenfeld S, et al. A phase I/II trial of pazopanib in combination with lapatinib in adult patients with relapsed malignant glioma. Clin Cancer Res. 2013;19:900–8.View ArticlePubMedGoogle Scholar
- Polak JM, Bloom SR. The central and peripheral distribution of neurotensin. Ann New York Acad Sci. 1982;400:75–93.View ArticleGoogle Scholar
- Cooper PE, Fernstrom MH, Rorstad OP, Leeman SE, Martin JB. The regional distribution of somatostatin, substance P and neurotensin in human brain. Brain Res. 1981;218:219–32.View ArticlePubMedGoogle Scholar
- Evers BM. Neurotensin and growth of normal and neoplastic tissues. Peptides. 2006;27:2424–33.6.View ArticlePubMedGoogle Scholar
- Dupouy S, Mourra N, Doan VK, Gompel A, Alifano M, Forgez P. The potential use of the neurotensin high affinity receptor 1 as a biomarker for cancer progression and as a component of personalized medicine in selective cancers. Biochimie. 2011;93:1369–78.View ArticlePubMedGoogle Scholar
- Alifano M, Loi M, Camilleri-Broet S, Dupouy S, Régnard JF, Forgez P. Neurotensin expression and outcome of malignant pleural mesothelioma. Biochimie. 2010;92:164–70.View ArticlePubMedGoogle Scholar
- Yi L, Xiao H, Xu M, Ye X, Hu J, Li F, et al. Glioma-initiating cells: a predominant role in microglia/macrophages tropism to glioma. J Neuroimmunol. 2011;232:75–82.View ArticlePubMedGoogle Scholar
- Sun L, Hui AM, Su Q, Vortmeyer A, Kotliarov Y, Pastorino S, et al. Neuronal and glioma-derived stem cell factor induces angiogenesis within the brain. Cancer Cell. 2006;9:287–300.View ArticlePubMedGoogle Scholar
- Gravendeel LA, Kouwenhoven MC, Gevaert O, de Rooi JJ, Stubbs AP, Duijm JE, et al. Intrinsic gene expression profiles of gliomas are a better predictor of survival than histology. Cancer Res. 2009;69:9065–72.View ArticlePubMedGoogle Scholar
- Kitabgi P, Checler F, Mazella J, Vincent JP. Pharmacology and biochemistry of neurotensin receptors. Rev Clin Basic Pharm. 1985;5:397–486.PubMedGoogle Scholar
- Vincent JP. Neurotensin receptors: binding properties, transduction pathways, and structure. Cell Mol Neurobiol. 1995;15:501–12.View ArticlePubMedGoogle Scholar
- Vincent JP, Mazella J, Kitabgi P. Neurotensin and neurotensin receptors. Trends Pharmacol Sci. 1999;20:302–9.View ArticlePubMedGoogle Scholar
- Hermans E, Maloteaux JM. Mechanisms of regulation of neurotensin receptors. Pharmacol Ther. 1998;79:89–104.View ArticlePubMedGoogle Scholar
- Demont Y, Corbet C, Page A, Ataman-Onal Y, Choquet-Kastylevsky G, Fliniaux I, et al. Pro-nerve growth factor induces autocrine stimulation of breast cancer cell invasion through tropomyosin-related kinase A (TrkA) and sortilin protein. J Biol Chem. 2012;287:1923–31.View ArticlePubMed CentralPubMedGoogle Scholar
- Souaze F, Dupouy S, Viardot-Foucault V, Bruyneel E, Attoub S, Gespach C, et al. Expression of neurotensin and NT1 receptor in human breast cancer: a potential role in tumor progression. Cancer Res. 2006;66:6243–9.View ArticlePubMedGoogle Scholar
- Tang KH, Ma S, Lee TK, Chan YP, Kwan PS, Tong CM, et al. CD133(+) liver tumor-initiating cells promote tumor angiogenesis, growth, and self-renewal through neurotensin/interleukin-8/CXCL1 signaling. Hepatology. 2012;55:807–20.View ArticlePubMedGoogle Scholar
- Massa F, Tormo A, Beraud-Dufour S, Coppola T, Mazella J. Neurotensin-induced Erk1/2 phosphorylation and growth of human colonic cancer cells are independent from growth factors receptors activation. Biochem Biophys Res Commun. 2011;414:118–22.View ArticlePubMedGoogle Scholar
- Bakirtzi K, Hatziapostolou M, Karagiannides I, Polytarchou C, Jaeger S, Iliopoulos D, et al. Neurotensin signaling activates microRNAs-21 and −155 and Akt, promotes tumor growth in mice, and is increased in human colon tumors. Gastroenterology. 2011;141:1749–61. e1741.View ArticlePubMedGoogle Scholar
- Mijatovic T, Gailly P, Mathieu V, De Neve N, Yeaton P, Kiss R, et al. Neurotensin is a versatile modulator of in vitro human pancreatic ductal adenocarcinoma cell (PDAC) migration. Cell Oncol. 2007;29:315–26.PubMedGoogle Scholar
- Coppola T, Beraud-Dufour S, Antoine A, Vincent JP, Mazella J. Neurotensin protects pancreatic beta cells from apoptosis. Int J Biochem Cell Biol. 2008;40:2296–302.View ArticlePubMedGoogle Scholar
- Yu SC, Bian XW. Enrichment of cancer stem cells based on heterogeneity of invasiveness. Stem Cell Rev. 2009;5:66–71.View ArticlePubMedGoogle Scholar
- Mantovani A, Allavena P, Sica A, Balkwill F. Cancer-related inflammation. Nature. 2008;454:436–44.View ArticlePubMedGoogle Scholar
- Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell. 2010;140:883–99.View ArticlePubMed CentralPubMedGoogle Scholar
- Watters JJ, Schartner JM, Badie B. Microglia function in brain tumors. J Neurosci Res. 2005;81:447–55.View ArticlePubMedGoogle Scholar
- Martin S, Dicou E, Vincent JP, Mazella J. Neurotensin and the neurotensin receptor-3 in microglial cells. J Neurosci Res. 2005;81:322–6.View ArticlePubMedGoogle Scholar
- Lewis CE, Pollard JW. Distinct role of macrophages in different tumor microenvironments. Cancer Res. 2006;66:605–12.View ArticlePubMedGoogle Scholar
- Olszewski U, Hamilton G. Neurotensin signaling induces intracellular alkalinization and interleukin-8 expression in human pancreatic cancer cells. Mol Oncol. 2009;3:204–13.View ArticlePubMedGoogle Scholar
- Yu J, Ren X, Chen Y, Liu P, Wei X, Li H, et al. Dysfunctional activation of neurotensin/IL-8 pathway in hepatocellular carcinoma is associated with increased inflammatory response in microenvironment, more epithelial mesenchymal transition in cancer and worse prognosis in patients. PLoS One. 2013;8:e56069.View ArticlePubMed CentralPubMedGoogle Scholar
- Wang X, Wang Q, Ives KL, Evers BM. Curcumin inhibits neurotensin-mediated interleukin-8 production and migration of HCT116 human colon cancer cells. Clin Cancer Res. 2006;12:5346–55.View ArticlePubMed CentralPubMedGoogle Scholar
- Waugh DJ, Wilson C. The interleukin-8 pathway in cancer. Clin Cancer Res. 2008;14:6735–41.View ArticlePubMedGoogle Scholar
- Zhao D, Zhan Y, Zeng H, Koon HW, Moyer MP, Pothoulakis C. Neurotensin stimulates expression of early growth response gene-1 and EGF receptor through MAP kinase activation in human colonic epithelial cells. Int J Cancer. 2007;120:1652–6.View ArticlePubMed CentralPubMedGoogle Scholar
- Kisfalvi K, Guha S, Rozengurt E. Neurotensin and EGF induce synergistic stimulation of DNA synthesis by increasing the duration of ERK signaling in ductal pancreatic cancer cells. J Cell Physiol. 2005;202:880–90.View ArticlePubMedGoogle Scholar
- Guha S, Lunn JA, Santiskulvong C, Rozengurt E. Neurotensin stimulates protein kinase C-dependent mitogenic signaling in human pancreatic carcinoma cell line PANC-1. Cancer Res. 2003;63:2379–87.PubMedGoogle Scholar
- Kisfalvi K, Hurd C, Guha S, Rozengurt E. Induced overexpression of protein kinase D1 stimulates mitogenic signaling in human pancreatic carcinoma PANC-1 cells. J Cell Physiol. 2010;223:309–16.PubMed CentralPubMedGoogle Scholar
- Hassan S, Dobner PR, Carraway RE. Involvement of MAP-kinase, PI3-kinase and EGF-receptor in the stimulatory effect of Neurotensin on DNA synthesis in PC3 cells. Regul Pept. 2004;120:155–66.View ArticlePubMedGoogle Scholar
- Dupouy S, Doan VK, Wu Z, Mourra N, Liu J, De Wever O, et al. Activation of EGFR, HER2 and HER3 by neurotensin/neurotensin receptor 1 renders breast tumors aggressive yet highly responsive to lapatinib and metformin in mice. Oncotarget. 2014;30:8235–51.Google Scholar
- Younes M, Wu ZS, Lupo AM, Mourra N, Takahashi T, Fléjou JF, et al. Neurotensin (NTS) and its receptor (NTSR1) causes EGFR, HER2 and HER3 over-expression and their autocrine/paracrine activation in lung tumors, confirming responsiveness to erlotinib. Oncotarget. 2014;5(18):8252–69.PubMed CentralPubMedGoogle Scholar
- Amorino GP, Deeble PD, Parsons SJ. Neurotensin stimulates mitogenesis of prostate cancer cells through a novel c-Src/Stat5b pathway. Oncogene. 2007;26:745–56.View ArticlePubMedGoogle Scholar
- Feng H, Hu B, Vuori K, Sarkaria JN, Furnari FB, Cavenee WK, et al. EGFRvIII stimulates glioma growth and invasion through PKA-dependent serine phosphorylation of Dock180. Oncogene. 2014;33:2504–12.View ArticlePubMedGoogle Scholar
- Sintupisut N, Liu PL, Yeang CH. An integrative characterization of recurrent molecular aberrations in glioblastoma genomes. Nucleic Acids Res. 2013;41:8803–21.View ArticlePubMed CentralPubMedGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.