Circulating T regulatory cells migration and phenotype in glioblastoma patients: an in vitro study

Glioblastoma multiforme (GBM) is the most aggressive primary human brain tumor. The relatively high amount of T regulatory lymphocytes present in the tumor, contributes to the establishment of an immunosuppressive microenvironment. Samples of peripheral blood were collected from GBM patients and healthy controls and a purified population of Treg (CD4⁺/CD25^bright) was isolated using flow cytometric cell sorting. Treg migrating capacities toward human glioma cell line conditioned medium were evaluated through an in vitro migration test. Our data show that supernatants collected from GBM cell lines were more attractant to Treg when compared to complete standard medium. The addition of an anti-CCL2 antibody to conditioned medium decreased conditioned medium-depending Treg migration, suggesting that CCL2 (also known as Monocyte Chemoattractant Protein, MCP-1) is implicated in the process. The number of circulating CD4⁺/μL or Treg/μL was similar in GBM patients and controls. Specific Treg markers (FOXP3; CD127; Helios; GITR; CTLA4; CD95; CCR2, CCR4; CCR7) were screened in peripheral blood and no differences could be detected between the two populations. These data confirm that the tumor microenvironment is attractive to Treg, which tend to migrate toward the tumor region changing the immunological response. Though we provide evidence that CCL2 is implicated in Treg migration, other factors are needed as well to provide such effect.     

Ethanol promotes mammary tumor growth and angiogenesis: the involvement of chemoattractant factor MCP-1

Alcohol consumption is a risk factor for breast cancer in humans. Experimental studies indicate that alcohol exposure promotes malignant progression of mammary tumors. However, the underlying cellular and molecular mechanisms remain unclear. Alcohol induces a pro-inflammatory response by modulating the expression of cytokines and chemokines. Monocyte chemoattractant protein-1 (MCP-1), also known as chemokine (C-C motif) ligand 2, is a pro-inflammatory chemokine implicated in breast cancer development/malignancy. We investigated the role of MCP-1 in alcohol-promoted mammary tumor progression. Using a xenograft model, we demonstrated that alcohol increased tumor angiogenesis and promoted growth/metastasis of breast cancer cells in C57BL/6 mice. Alcohol up-regulated the expression of MCP-1 and its receptor CCR2 in breast cancer cells in vitro and in vivo. Using a three-dimensional tumor/endothelial cell co-culture system, we demonstrated MCP-1 regulated tumor/endothelial cell interaction and promoted tumor angiogenesis. More importantly, MCP-1 mediated alcohol-promoted angiogenesis; an antagonist of the MCP-1 receptor CCR2 significantly inhibited alcohol-stimulated tumor angiogenesis. The CCR2 antagonist abolished ethanol-stimulated growth of mammary tumors in mice. We further demonstrated that MCP-1 enhanced the migration, but not the proliferation of endothelial cells as well as breast cancer cells. These results suggest that MCP-1 plays an important role in ethanol-stimulated tumor angiogenesis and tumor progression.     

Exogenous endothelin-1 induces cell migration and matrix metalloproteinase expression in U251 human glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and lethal type of primary brain tumor characterized by its rapid infiltration to surrounding tissues during the early stages. The fast spreading of GBM obscures the initiation of the tumor mass making the treatment outcome undesirable. Endothelin-1 is known as a secretory protein presented in various types of brain cells, which has been indicated as a factor for cancer pathology. The aim of the present study was to investigate the molecular mechanism of cell migration in GBM. We found that various malignant glioma cells expressed higher amounts of endothelin-1, ET_A, and ET_B receptors than nonmalignant human astrocytes. The application of endothelin-1 enhanced the migratory activity in human U251 glioma cells corresponding to increased expression of matrix metalloproteinase (MMP)-9 and MMP-13. The endothelin-1-induced cell migration was attenuated by MMP-9 and MMP-13 inhibitors and inhibitors of mitogen-activated protein (MAP) kinase and PI3 kinase/Akt. Furthermore, the elevated levels of phosphate c-Jun accumulation in the nucleus and activator protein-1 (AP-1)-DNA binding activity were also found in endothelin-1 treated glioma cells. In migration-prone sublines, cells with greater migration ability showed higher endothelin-1, ET_B receptor, and MMP expressions. These results indicate that endothelin-1 activates MAP kinase and AP-1 signaling, resulting in enhanced MMP-9 and MMP-13 expressions and cell migration in GBM.     

The phosphorylation of ephrin‐B2 ligand promotes glioma cell migration and invasion

To reveal molecular drivers of glioma invasion, two distinct glioblastoma (GBM) cell phenotypes (invading cells and tumor core cells) were collected from 19 GBM specimens using laser capture microdissection. Isolated RNA underwent whole human genome expression profiling to identify differentially expressed genes. Pathway enrichment analysis highlighted the bidirectional receptor/ligand tyrosine kinase system, EphB/ephrin‐B, as the most tightly linked system to the invading cell phenotype. Clinical relevance of ephrin‐B genes was confirmed in a clinically annotated expression data set of 195 brain biopsy specimens. Levels of ephrin‐B1 and ‐B2 mRNA were significantly higher in GBM (n = 82) than in normal brain (n = 24). Kaplan–Meier analysis demonstrated ephrin‐B2, but not ephrin‐B1, expression levels were significantly associated with short term survival in malignant astrocytomas (n = 97, p = 0.016). In human brain tumor specimens, the production and phosphorylation of ephrin‐B2 were high in GBM. Immunohistochemistry demonstrated ephrin‐B2 localization primarily in GBM cells but not in normal brain. A highly invasive glioma cell line, U87, expressed high levels of ephrin‐B2 compared with relatively less invasive cell lines. Treatment with EphB2/Fc chimera further enhanced migration and invasion of U87 cells, whereas treatment with an ephrin‐B2 blocking antibody significantly slowed migration and invasion. Forced expression of ephrin‐B2 in the U251 cell line stimulated migration and invasion in vitro and ex vivo, concomitant with tyrosine phosphorylation of ephrin‐B2. These results demonstrate that high expression of ephrin‐B2 is a strong predictor of short‐term survival and that ephrin‐B2 plays a critical role in glioma invasion rendering this signaling pathway as a potential therapeutic target.     

NHERF-1: Modulator of Glioblastoma Cell Migration and Invasion,

The invasive nature of malignant gliomas is a clinical problem rendering tumors incurable by conventional treatment modalities such as surgery, ionizing radiation, and temozolomide. Na⁺/H⁺ exchanger regulatory factor 1 (NHERF-1) is a multifunctional adaptor protein, recruiting cytoplasmic signaling proteins and membrane receptors/transporters into functional complexes. This study revealed that NHERF-1 expression is increased in highly invasive cells that reside in the rim of glioblastoma multiforme (GBM) tumors and that NHERF-1 sustains glioma migration and invasion. Gene expression profiles were evaluated from laser capture-microdissected human GBM cells isolated from patient tumor cores and corresponding invaded white matter regions. The role of NHERF-1 in the migration and dispersion of GBM cell lines was examined by reducing its expression with small-interfering RNA followed by radial migration, three-dimensional collagen dispersion, immunofluorescence, and survival assays. The in situ expression of NHERF-1 protein was restricted to glioma cells and the vascular endothelium, with minimal to no detection in adjacent normal brain tissue. Depletion of NHERF-1 arrested migration and dispersion of glioma cell lines and caused an increase in cell-cell cohesiveness. Glioblastoma multiforme cells with depleted NHERF-1 evidenced a marked decrease in stress fibers, a larger cell size, and a more rounded shape with fewer cellular processes. When NHERF-1 expression was reduced, glioma cells became sensitized to temozolomide treatment resulting in increased apoptosis. Taken together, these results provide the first evidence for NHERF-1 as a participant in the highly invasive phenotype of malignant gliomas and implicate NHERF-1 as a possible therapeutic target for treatment of GBM.     

Vitamin D receptor expression is associated with improved overall survival in human glioblastoma multiforme

Vitamin D and its analogs have been shown to display anti-proliferative effects in a wide variety of cancer types including glioblastoma multiforme (GBM). These anticancer effects are mediated by its active metabolite, 1α, 25-dihydroxyvitamin D₃ (calcitriol) acting mainly through vitamin D receptor (VDR) signaling. In addition to its involvement in calcitriol action, VDR has also been demonstrated to be useful as a prognostic factor for some types of cancer. However, to our knowledge, there are no studies evaluating the expression of VDR protein and its association with outcome in gliomas. Therefore, we investigated VDR expression by using immunohistochemical analysis in human glioma tissue microarrays, and analyzed the association between VDR expression and clinico-pathological parameters. We further investigated the effects of genetic and pharmacologic modulation of VDR on survival and migration of glioma cell lines. Our data demonstrate that VDR is increased in tumor tissues when compared with VDR in non-malignant brains, and that VDR expression is associated with an improved outcome in patients with GBM. We also show that both genetic and pharmacologic modulation of VDR modulates GBM cellular migration and survival and that VDR is necessary for calcitriol-mediated effects on migration. Altogether these results provide some limited evidence supporting a role for VDR in glioma progression.     

Role of glycosyltransferase PomGnT1 in glioblastoma progression

BackgroundGlioblastoma multiforme (GBM) is the most aggressive and invasive brain tumor, for which novel prognostic markers and predictors of therapeutic response are urgently needed. We reported previously that levels of peptide-O-linked mannose β-1,2-N-acetylglucosaminyltransferase 1 (PomGnT1) in glioma specimens correlated with tumor grade. However, the prognostic significance of PomGnT1 in glioma patients and its function in GBM progression remain unknown.MethodsClinical relevance of PomGnT1 in GBM patients'' prognosis was analyzed both in a clinically annotated expression dataset of 446 GBM tumor specimens and in 82 GBM tumor samples collected at our institution. The function of PomGnT1 in glioma growth and invasion, and the underlying mechanisms of PomGnT1 regulation were explored in vitro and in vivo.ResultsPomGnT1 expression in GBM tissues was closely associated with poor prognosis in GBM patients. Forced overexpression of PomGnT1 in glioblastoma cells impaired cell adhesion and increased their proliferation and invasion in vitro. Subsequent in vivo experiments showed that overexpression of PomGnT1 promoted tumor growth and shortened the survival time of tumor-bearing mice in an orthotopic model. Conversely, stable short hairpin RNA–mediated knockdown of PomGnT1 expression produced opposite effects both in vitro and in vivo. Mechanistic studies revealed that activation of epidermal growth factor receptor (EGFR) resulted in EGFR/extracellular signal-regulated kinase–dependent upregulation of PomGnT1, downregulation of receptor-type protein tyrosine phosphatase β, and activation of β-catenin pathway signaling.ConclusionThese findings suggest that PomGnT1 promotes GBM progression via activation of β-catenin and may serve as a prognostic factor for glioma patient survival as well as a novel molecular target for anticancer therapy in malignant glioma.     

SRC抑制胶质母细胞瘤的增殖、迁移、侵袭和干性维持的分子机制

目的 研究SRC在胶质母细胞瘤发生发展中的作用,并初步探讨可能的分子机制。方法  采用生物信息学的方法分析SRC在胶质母细胞瘤中的表达变化;利用shRNA下调胶质瘤母细胞系U87MG中SRC的表达,通过RT-PCR和免疫印迹法验证其抑制效率,并筛选出稳定干涉的细胞株;采用WST-1法、划痕愈合实验和Transwell迁移实验检测SRC shRNA干涉后细胞增殖、迁移和侵袭能力的变化;利用干细胞培养液筛选出SRC shRNA稳定干涉的胶质瘤干细胞,观察SRC shRNA对肿瘤干细胞干性的影响;利用细胞免疫荧光法观察干性基因SOX2的表达变化。结果 在胶质母细胞瘤标本中SRC的表达水平高于对照组,筛选到两条有效的SRC shRNA序列;通过shRNA下调SRC的表达后可以显著抑制胶质瘤母细胞U87MG的增殖、迁移、侵袭和肿瘤干细胞干性维持,并且可以明显抑制SOX2的表达。结论 SRC通过调控胶质母细胞瘤的增殖、迁移、侵袭和干性维持影响其发生发展,其对干性维持的作用可能是通过影响SOX2的表达实现的。     

Activation of hypoxia signaling induces phenotypic transformation of glioma cells: implications for bevacizumab antiangiogenic therapy

Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), can attenuate tumor-associated edema and improve patient symptoms but based on magnetic resonance imaging, is associated with non-enhancing tumor progression and possibly gliosarcoma differentiation. To gain insight into these findings, we investigated the role of hypoxia and epithelial-mesenchymal transition (EMT)-associated proteins in GBM. Tumor markers of hypoxia and EMT were upregulated in bevacizumab-treated tumors from GBM patients compared to untreated counterparts. Exposure of glioma cells to 1% oxygen tension increased cell proliferation, expression of EMT-associated proteins and enhanced cell migration in vitro. These phenotypic changes were significantly attenuated by pharmacologic knockdown of hypoxia-inducible Factor 1α (HIF1α) or HIF2α, indicating that HIFs represent a therapeutic target for mesenchymal GBM cells. These findings provide insights into potential development of novel therapeutic targeting of angiogenesis-specific pathways in GBM.     

Global analysis of H3K4me3 and H3K27me3 profiles in glioblastoma stem cells and identification of SLC17A7 as a bivalent tumor suppressor gene

Epigenetic changes, including H3K4me3 and H3K27me3 histone modification, play an important role in carcinogenesis. However, no genome-wide histone modification map has been generated for gliomas. Here, we report a genome-wide map of H3K4me3 and H3K27me3 histone modifications for 8 glioma stem cell (GSC) lines, together with the associated gene activation or repression patterns. In addition, we compared the genome-wide histone modification maps of GSC lines to those of astrocytes to identify unique gene activation or repression profiles in GSCs and astrocytes. We also identified a set of bivalent genes, which are genes that are associated with both H3K4me3 and H3K27me3 marks and are poised for action in embryonic stem cells. These bivalent genes are potential targets for inducing differentiation in glioblastoma (GBM) as a therapeutic approach. Finally, we identified SLC17A7 as a bivalent tumor suppressor gene in GBM, as it is down-regulated at both the protein and RNA levels in GBM tissues compared with normal brain tissues, and it inhibits GBM cell proliferation, migration and invasion.     

趋化因子受体MCP-1/CCR2在多发性骨髓瘤中基质细胞与瘤细胞的异常表达

 [摘要] 目的 观察趋化因子（MCP-1）及其趋化因子受体（CCR2）在多发性骨髓瘤（MM）患者骨髓单个细胞,骨髓基质细胞中的表达。方法 选择2011年5月—2011年9月山西医科大学第一附属医院血液科住院患者,15例经国内统一标准确诊为多发性骨髓瘤的初发病人,10例为一般血液病患者。①实验方法,取样本进行骨髓瘤细胞与骨髓基质细胞的分离培养,在含13%小牛血清的RPMI1640培养液中培养,每2天更换1次培养液,本实验用的细胞均处于对数生长期。②实验评估,采用流式细胞术检测骨髓瘤患者骨髓瘤细胞,骨髓基质细胞中MCP-1、CCR2。结果 实验评估方法显示约2/3的多发性骨髓瘤患者骨髓均表达MCP-1、CCR2,而对照组的骨髓中趋化因子MCP-1、CCR2基本无表达。结论 大部分多发性骨髓瘤患者骨髓均表达MCP-1、CCR2。 MCP-1及其特异性受体CCR2是MM细胞表面表达的主要趋化因子,对MM的疾病的进展起着一个比较重要的作用。     

A pivotal role for CC chemokine receptor 5 in T-cell migration to tumor sites induced by interleukin 12 treatment in tumor-bearing mice

Interleukin (IL) 12 treatment in the CSA1M and OV-HM, but not in Meth A tumor models,induces tumor regression that is associated with T-cell migration to tumor sites.Here, we investigated the role of the CC chemokine receptor (CCR)5 in T-cell migration induced after IL-12 treatment. In the two IL-12-responsive tumor models (CSA1M and OV-HM), IL-12 treatment up-regulated the mRNA expression of CCR5 in splenic T cells as well as ligands for CCR5, such as macrophage inflammatory protein (MIP) 1alpha and MIP-1beta in tumor masses. In contrast, the expression of CCR5 in spleens and MIP-1alpha/MIP-1beta in tumor masses was marginally induced before and even after IL-12 treatment in the Meth A model in which T-cell migration is not observed. T cells infiltrating tumor masses in the former two IL-12-responsive models expressed CCR5. Administration of a synthetic CCR5 antagonist TAK-779 to tumor-bearing mice during IL-12 immunotherapy prevented T-cell migration and tumor regression. Furthermore, anti-CCR5 antibody was found to inhibit T-cell migration in the lymphoid cell migration assay. Namely, although splenic T cells prepared from IL-12-treated CSA1M or OV-HM-bearing mice migrated into the corresponding tumor masses in recipient mice, the migration was inhibited when donor T cells were treated with anti-CCR5 antibody before the injection. These results indicate a critical role for CCR5 in the induction of T-cell migration to tumor sites after IL-12 treatment.     

High expression of L-type amino acid transporter 1 in infiltrating glioma cells

L-type amino acid transporter 1 (LAT1), a neutral amino acid transport agent, is essential for the transport of large neutral amino acids. LAT1 also corresponds to tumor-associated gene-1 (TA1), an oncofetal antigen that is expressed primarily in fetal tissues and cancer cells such as glioma cells. We have investigated the expression of the transporter in the human primary glioma tissue from 68 patients. Among these patients, we could see the border zone between tumors and normal bain tissues in 10 patients. By WHO criteria, two of the specimens were diagnosed as grade 2, three as grade 3, and five as grade 4 [glioblastoma multiforme (GBM)]. In 9 of 10 cases, we could identify the infiltrating glioma cells associated with stronger immunoreactivity for LAT1. These tumor cells aggregated around the neurons in the border zone and were often found in the perivascular space. In one GBM case, the tumors seemed to develop expansively and separated from the normal brain with a border of arachnoid membrane. The expression of LAT1 was always higher in infiltrating glioma cells than in cells located in the center of the tumor. These findings suggest that LAT1 is one of the molecular targets for glioma therapy.     

RasGRP3 regulates the migration of glioma cells via interaction with Arp3

Glioblastoma (GBM), the most aggressive primary brain tumors, are highly infiltrative. Although GBM express high Ras activity and Ras proteins have been implicated in gliomagenesis, Ras-activating mutations are not frequent in these tumors. RasGRP3, an important signaling protein responsive to diacylglycerol (DAG), increases Ras activation. Here, we examined the expression and functions of RasGRP3 in GBM and glioma cells. RasGRP3 expression was upregulated in GBM specimens and glioma stem cells compared with normal brains and neural stem cells, respectively. RasGRP3 activated Ras and Rap1 in glioma cells and increased cell migration and invasion partially via Ras activation. Using pull-down assay and mass spectroscopy we identified the actin-related protein, Arp3, as a novel interacting protein of RasGRP3. The interaction of RasGRP3 and Arp3 was validated by immunofluorescence staining and co-immunoprecipitation, and PMA, which activates RasGRP3 and induces its translocation to the peri-nuclear region, increased the association of Arp3 and RasGRP3. Arp3 was upregulated in GBM, regulated cell spreading and migration and its silencing partially decreased these effects of RasGRP3 in glioma cells. In summary, RasGRP3 acts as an important integrating signaling protein of the DAG and Ras signaling pathways and actin polymerization and represents an important therapeutic target in GBM.