Invasion Of Malignant Gliomas Research Articles

Overexpression of hyaluronan-binding protein 1 promotes the proliferation, migration, and invasion of malignant glioma.

BackgroundHyaluronan binding protein 1 (HABP1) is a member of the hyaladherin family and is involved in T cell activation. It is a high-grade polysaccharide. The CD44 family is also a member of this family. It is expressed in many tumor cells at higher levels than the corresponding normal tissues. It is related to the occurrence and development of tumor cells, invasiveness, and lymph node metastasis. However, the mechanism of HABP1 in glioma remains unclear.MethodsWe focused on the proliferation and invasion of HABP1 in gliomas. The expression of HABP1 was firstly investigated by immunohistochemical staining and by comparing different grades of glioma tissues from 80 patients with glioma. Then, the influence of migration and proliferation through different human glioma cell line models were investigated.ResultsBy collecting a large number of clinical pathological tissues and patient data, it was found that HABP1 expression was significantly correlated with glioma, and gels with a high expression of HABP1 and a low expression of HABP1 were screened by different glioma cell lines. In the cytomorphological analysis, short interfering RNA with HABP1 significantly inhibited the proliferation of glioma, and the overexpression of HABP1 enhanced glioma invasion and migration.ConclusionsHABP1 is likely to become a new targeting factor for the treatment of glioma. The mechanism of action of HABP1 in glioma remains to be further studied.

EXTH-70. ENHANCEMENT OF ANTITUMOR ACTIVITY BY USING 5-ALA–MEDIATED SONODYNAMIC THERAPY TO INDUCE APOPTOSIS AND NECROSIS IN A MOUSE GLIOMA MODEL

Abstract OBJECTIVE High invasiveness of malignant gliomas frequently causes local tumor recurrence. To control such recurrence, novel therapies targeted toward infiltrating glioma cells are required. Here, we examined cytotoxic effects of sonodynamic therapy (SDT) combined with a sonosensitizer, 5-aminolevulinic acid (5-ALA), on malignant gliomas both in vitro and in vivo. METHODS In vitro cytotoxicity of 5-ALA-SDT was evaluated in U87 and U251 glioma cells and in U251Oct-3/4 glioma stemlike cells. Treatment-related apoptosis was analyzed using flow cytometry. Intracellular reactive oxygen species (ROS) were measured and the role of ROS in treatment-related cytotoxicity was examined. Effects of 5-ALA-SDT with high-intensity focused ultrasound (HIFU) on tumor growth, survival of glioma-transplanted mice, and histological features of the mouse brains were investigated. RESULTS The 5-ALA-SDT inhibited cell growth and changed cell morphology. Flow cytometric analysis indicated that 5-ALA-SDT induced apoptotic cell death. The 5-ALA-SDT generated higher ROS than in the control group, and inhibition of ROS generation completely eliminated the cytotoxic effects of 5-ALA-SDT. In the in vivo study, 5-ALA-SDT with HIFU greatly prolonged survival of the tumor-bearing mice compared with that of the control group (p < 0.05). Histologically, 5-ALA-SDT produced mainly necrosis of the tumor tissue in the focus area and induced apoptosis of the tumor cells in the perifocus area around the target of the HIFU-irradiated field. Normal brain tissues around the ultrasonic irradiation field of HIFU remained intact. CONCLUSIONS The 5-ALA-SDT was cytotoxic toward malignant gliomas. Generation of ROS by the SDT was thought to promote apoptosis of glioma cells. The 5-ALA-SDT with HIFU induced tumor necrosis in the focus area and apoptosis in the perifocus area of the HIFU-irradiated field. These results suggest that 5-ALA-SDT with HIFU may present a less invasive and tumor-specific therapy, not only for a tumor mass but also for infiltrating tumor cells in malignant gliomas.

Enhancement of antitumor activity by using 5-ALA-mediated sonodynamic therapy to induce apoptosis in malignant gliomas: significance of high-intensity focused ultrasound on 5-ALA-SDT in a mouse glioma model.

OBJECTIVEHigh invasiveness of malignant gliomas frequently causes early local recurrence of the tumor, resulting in extremely poor outcome. To control such recurrence, novel therapies targeted toward infiltrating glioma cells around the tumor border are required. Here, the authors investigated the antitumor activity of sonodynamic therapy (SDT) combined with a sonosensitizer, 5-aminolevulinic acid (5-ALA), on malignant gliomas to explore the possibility for clinical use of 5-ALA-mediated SDT (5-ALA-SDT).METHODSIn vitro cytotoxicity of 5-ALA-SDT was evaluated in U87 and U251 glioma cells and in U251Oct-3/4 glioma stemlike cells. Treatment-related apoptosis was analyzed using flow cytometry and TUNEL staining. Intracellular reactive oxygen species (ROS) were measured and the role of ROS in treatment-related cytotoxicity was examined by analysis of the effect of pretreatment with the radical scavenger edaravone. Effects of 5-ALA-SDT with high-intensity focused ultrasound (HIFU) on tumor growth, survival of glioma-transplanted mice, and histological features of the mouse brains were investigated.RESULTSThe 5-ALA-SDT inhibited cell growth and changed cell morphology, inducing cell shrinkage, vacuolization, and swelling. Flow cytometric analysis and TUNEL staining indicated that 5-ALA-SDT induced apoptotic cell death in all gliomas. The 5-ALA-SDT generated significantly higher ROS than in the control group, and inhibition of ROS generation by edaravone completely eliminated the cytotoxic effects of 5-ALA-SDT. In the in vivo study, 5-ALA-SDT with HIFU greatly prolonged survival of the tumor-bearing mice compared with that of the control group (p < 0.05). Histologically, 5-ALA-SDT produced mainly necrosis of the tumor tissue in the focus area and induced apoptosis of the tumor cells in the perifocus area around the target of the HIFU-irradiated field. The proliferative activity of the entire tumor was markedly decreased. Normal brain tissues around the ultrasonic irradiation field of HIFU remained intact.CONCLUSIONSThe 5-ALA-SDT was cytotoxic toward malignant gliomas. Generation of ROS by the SDT was thought to promote apoptosis of glioma cells. The 5-ALA-SDT with HIFU induced tumor necrosis in the focus area and apoptosis in the perifocus area of the HIFU-irradiated field, whereas the surrounding brain tissue remained normal, resulting in longer survival of the HIFU-treated mice compared with that of untreated mice. These results suggest that 5-ALA-SDT with HIFU may present a less invasive and tumor-specific therapy, not only for a tumor mass but also for infiltrating tumor cells in malignant gliomas.

Epidermal Growth Factor (EGF) Promotes Human Malignant Glioma Invasion by Mediating Secretion of Human Cytomegalovirus Infected Monocyte-Derived Macrophages

Human malignant glioma is the most aggressive brain tumor which lacks efficient therapies. Accumulating evidence indicates that human malignant gliomas are universally infected with human cytomegalovirus (HCMV). Recent studies demonstrate that tumor-associated macrophages (TAMs) density is associated with glioma grade. We hypothesize that virus affected the secretion of macrophages which infiltrated into glioma to promote the glioma cell invasion. Supporting this hypothesis, we showed that the secretion factor EGF had increased when HCMV infect macrophages. And the expression of the epidermal growth factor receptor (EGFR) on the surface of malignant U87 cells also up-regulated, indicating the infection of HCMV can impact the secretion factor EGF of macrophages and then activates EGFR of malignant glioma cells. We measured focal adhesion kinase FAK Tyr397 which is necessary for cell mobility. HCMV infected-macrophages can up-regulate the expression of FAK Tyr397 of malignant U87 cells. Together, these results provide evidences that EGF from infected-macrophage can activate EGFR of human malignant glioma and promote glioma cell invasion. In conclusion, these findings indicate that infected-macrophage with HCMV play an important role in the invasion of glioma and may act as a potential target to prevent its invasion.

Inhibition of glioblastoma cell invasion by hsa-miR-145-5p and hsa-miR-31-5p co-overexpression in human mesenchymal stem cells.

Human bone marrow–derived mesenchymal stem cells (hMSCs) show tropism for brain tumors and may be a useful vehicle for drug or gene delivery to malignant gliomas. Recently, some microRNAs (miRNAs) have been shown to suppress the invasiveness of malignant gliomas. To test their potential to become vehicles for the delivery of miRNA to malignant gliomas, hMSCs were engineered so that hMSC secretion of miRNAs that inhibit glioma cell invasion was enabled without altering the hMSC tropism for glioma cells. In coculture, hMSCs cotransfected with hsa-miR-145-5p and -31-5p miRNAs showed markedly reduced invasion by U87 glioma cells in a contact-dependent manner both in vitro and ex vivo, with invasion of hMSCs cotransfected with these 2 miRNAs by the U87 cells reduced to 60.7% compared with control cells. According to a Matrigel invasion assay, the tropism of the hMSCs for U87 cells was not affected. In glioma cell lines U251 and LN229, hMSCs exhibited tropism in vivo, and invasion of hMSCs cotransfected with hsa-miR-145-5p and -31-5p was also significantly less than that of control cells. When U87 cells were coimplanted into the striatum of organotypic rat brain slices with hMSCs cotransfected with hsa-miR-145 and -31-5p, the relative invasive area decreased by 37.1%; interestingly, these U87 cells showed a change to a rounded morphology that was apparent at the invasion front. Whole-genome microarray analysis of the expression levels of 58,341 genes revealed that the co-overexpression of hsa-miR-145-5p and -31-5p downregulated FSCN1 expression in U87 cells. This study demonstrates that miRNA overexpression in hMSCs can alter the function of glioma cells via contact-dependent transfer. Co-overexpression of multiple miRNAs may be a useful and novel therapeutic strategy. The study results suggest that hMSCs can be applied as a delivery vehicle for miRNAs.

Down-Regulation of AQP4 Expression via p38 MAPK Signaling in Temozolomide-Induced Glioma Cells Growth Inhibition and Invasion Impairment.

Glioma is the most common and lethal central nervous system tumors. Temozolomide (TMZ) is an effective drug for malignant glioma, however, the intracellular and molecular mechanisms behind this anti-cancer effect have yet to be fully understood. The aim of the present study was to determine whether TMZ inhibits proliferation, invasion of glioma cells in vitro and whether these effects can be mediated through modulation of aquaporin 4 (AQP4) and phosphorylation of the MAPK pathway. The viability of U87 and U251 human glioma cells was evaluated using MTT assay. The cell cycle distribution was detected with flow cytometry. Migration ability and invasion ability were tested by scratch assays and transwell assays, respectively. The levels of AQP4 and MAPK were measured using immunoblot analyses. Our results showed that TMZ inhibited proliferation, migration and invasion, and induced G2/M arrest in U87 and U251 glioma cell lines. These changes were associated with a decrease in the levels of AQP4 expression as well as activation phosphorylated level of p38. Treatment with a p38 chemical activator (anisomycin) resulted in similar effects as TMZ treatment on glioma cells. And p38 chemical inhibitor (SB203580) could block these effects in glioma treated with TMZ, suggesting a direct up-regulation of the p38 signaling pathway. Therefore, we identified that TMZ might have therapeutic potential for controlling proliferation, invasion of malignant glioma by inhibiting AQP4 expression through activation of p38 signal transduction pathway. J. Cell. Biochem. 118: 4905-4913, 2017. © 2017 Wiley Periodicals, Inc.

MiR-548b inhibits the proliferation and invasion of malignant gliomas by targeting metastasis tumor-associated protein-2.

microRNAs (miRNAs) play important roles in cancer development and progression. In this study, we explored the expression and biological roles of miR-548b in human gliomas. The expression of miR-548b in human glioma tissues and cell lines was examined. Gain-of-function experiments were conducted to determine the roles of miR-548b in glioma cell growth, invasiveness, and tumorigenesis. Bioinformatic analysis and luciferase reporter assays were performed to identify direct target genes for miR-548b. miR-548b was underexpressed in human glioma tissues and cell lines. Re-expression of miR-548b significantly inhibited the proliferation and colony formation of U87 and U373 glioma cells. Enforced expression of miR-548b significantly impaired the invasiveness of glioma cells. Notably, metastasis tumor-associated protein-2 (MTA2) was a direct target of miR-548b. Overexpression of miR-548b negatively regulated endogenous MTA2 expression in U87 cells. Rescue experiments with an MTA2 construct lacking the 3'-untranslated region showed that enforced expression of MTA2 significantly restored cell proliferation and invasion in miR-548b-overexpressing cells. In-vivo studies confirmed that miR-548b overexpression retarded the growth of U87 xenograft tumors, which was accompanied by reduced expression of MTA2. In conclusion, miR-548b exerts its tumor-suppressive activity in glioma through repression of MTA2. Restoration of miR-548b may have therapeutic potential in glioma.

Metallothinein 1E Enhances Glioma Invasion through Modulation Matrix Metalloproteinases-2 and 9 in U87MG Mouse Brain Tumor Model.

Malignant glioma cells invading surrounding normal brain are inoperable and resistant to radio- and chemotherapy, and eventually lead to tumor regrowth. Identification of genes related to motility is important for understanding the molecular biological behavior of invasive gliomas. According to our previous studies, Metallothionein 1E (MT1E) was identified to enhance migration of human malignant glioma cells. The purpose of this study was to confirm that MT1E could modulate glioma invasion in vivo. Firstly we established 2 cell lines; MTS23, overexpressed by MT1E complementary DNA construct and pV12 as control. The expression of matrix metalloproteinases (MMP)-2, -9 and a disintegrin and metalloproteinase 17 were increased in MTS23 compared with pV12. Furthermore it was confirmed that MT1E could modulate MMPs secretion and translocation of NFkB p50 and B-cell lymphoma-3 through small interfering ribonucleic acid knocked U87MG cells. Then MTS23 and pV12 were injected into intracranial region of 5 week old male nude mouse. After 4 weeks, for brain tissues of these two groups, histological analysis, and immunohistochemical stain of MMP-2, 9 and Nestin were performed. As results, the group injected with MTS23 showed irregular margin and tumor cells infiltrating the surrounding normal brain, while that of pV12 (control) had round and clear margin. And regrowth of tumor cells in MTS23 group was observed in another site apart from tumor cell inoculation. MT1E could enhance tumor proliferation and invasion of malignant glioma through regulation of activation and expression of MMPs.

Naringin inhibits the invasion and migration of human glioblastoma cell via downregulation of MMP-2 and MMP-9 expression and inactivation of p38 signaling pathway.

Gliomas are the most common and malignant primary brain tumors. They are associated with a poor prognosis despite the availability of multiple therapeutic options. Naringin, a common dietary flavonoid abundantly present in fruits and vegetables, is believed to possess strong anti-proliferative and anti-cancer properties. However, there are no reports describing its effects on the invasion and migration of glioblastoma cell lines. Our results showed that the treatment of U251 glioma cell lines with different concentrations of naringin inhibited the invasion and migration of these cells. In addition, we revealed a decrease in the levels of matrix metalloproteinases (MMP-2) and (MMP-9) expression as well as proteinase activity in U251 glioma cells. In contrast, the expression of tissue inhibitor of metalloproteinases (TIMP-1) and (TIMP-2) was increased. Furthermore, naringin treatment decreased significantly the phosphorylated level of p38. Combined treatment with a p38 inhibitor (SB203580) resulted in the synergistic reduction of MMP-2 and MMP-9 expressions correlated with an increase of TIMP-1 and TIMP-2 expressions and the anti-invasive properties. However, p38 chemical activator (anisomycin) could block these effects produced by naringin, suggesting a direct downregulation of the p38 signaling pathway. These data suggest that naringin may have therapeutic potential for controlling invasiveness of malignant gliomas by inhibiting of p38 signal transduction pathways.

Sublethal dose of irradiation enhances invasion of malignant glioma cells through p53-MMP 2 pathway in U87MG mouse brain tumor model.

BackgroundGlioblastoma is a highly lethal neoplasm that frequently recurs locally after radiotherapy, and most of these recurrences originate from near the irradiated target field. In the present study, we identified the effects of radiation on glioma invasion and p53, TIMP-2, and MMP-2 expression through in vitro and in vivo experiments.MethodsThe U87MG (wt p53) and U251 (mt p53) human malignant glioma cell lines were prepared, and the U2OS (wt 53) and Saos2 (del p53) osteosarcoma cell lines were used as p53 positive and negative controls. The four cell lines and p53 knock-downed U87MG cells received radiation (2–6 Gy) and were analyzed for expression of p53 and TIMP-2 by Western blot, and MMP-2 activity was detected by zymography. In addition, the effects of irradiation on directional invasion of malignant glioma were evaluated by implanting nude mice with bioluminescent u87-Fluc in vivo followed by MMP-2, p53, and TIMP-2 immunohisto-chemistry and in situ zymography.ResultsMMP-2 activity and p53 expression increased in proportional to the radiation dose in cell lines with wt p53, but not in the cell lines with del or mt p53. TIMP-2 expression did not increase in U87MG cells. MMP-2 activity decreased in p53 knock-downed U87MG cells but increased in the control group. Furthermore, radiation enhanced MMP-2 activity and increased tumor margin invasiveness in vivo. Tumor cells invaded by radiation overexpressed MMP-2 and p53 and revealed high gelatinolytic activity compared with those of non-radiated tumor cells.ConclusionRadiation-induced upregulation of p53 modulated MMP-2 activity, and the imbalance between MMP-2 and TIMP-2 may have an important role in glioblastoma invasion by degrading the extracellular matrix. Bioluminescent “U87-Fluc”was useful for observing tumor formation without sacrifice after implanting tumor cells in the mouse brain. These findings suggest that the radiotherapy involved field for malignant glioma needs to be reconsidered, and that future trials should investigate concurrent pharmacologic therapies that inhibit invasion associated with radiotherapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13014-015-0475-8) contains supplementary material, which is available to authorized users.

Hormones and immunity in cancer: are thyroid hormones endocrine players in the microglia/glioma cross-talk?

Accumulating evidence indicates that the endocrine and immune systems engage in complex cross-talks in which a prominent role is played by thyroid hormones (THs). The increase of resident vs. monocyte recruited macrophages was shown to be an important effector of the TH 3,3′,5′-Triiodo-L-thyronine (T3)-induced protection against inflammation and a key role of T3 in inhibiting the differentiation of peripheral monocytes into macrophages was observed. Herein, we report on the role of T3 as a modulator of microglia, the specialized macrophages of the central nervous system (CNS). Mounting evidence supports a role of microglia and macrophages in the growth and invasion of malignant glioma. In this respect, we unveil the putative involvement of T3 in the microglia/glioma cell communication. Since THs are known to cross the blood-brain barrier, we suggest that T3 not only exerts a direct modulation of brain cancer cell itself but also indirectly promotes glioma growth through a modulation of microglia. Our observations expand available information on the role of TH system in glioma and its microenvironment and highlight the endocrine modulation of microglia as an important target for future therapeutic development of glioma treatments.

Intermittent Induction of HIF-1α Produces Lasting Effects on Malignant Progression Independent of Its Continued Expression

Dysregulation of hypoxia-inducible transcription factors HIF-1α and HIF-2α correlates with poor prognosis in human cancers; yet, divergent and sometimes opposing activities of these factors in cancer biology have been observed. Adding to this complexity is that HIF-1α apparently possesses tumor-suppressing activities, as indicated by the loss-of-function mutations or even homozygous deletion of HIF1A in certain human cancers. As a step towards understanding this complexity, we employed 8-week intermittent induction of a stable HIF-1α variant, HIF1α(PP), in various cancer cell lines and examined the effects on malignant progression in xenografts of immunocompromised mice in comparison to those of HIF2α(PP). Although 8-week treatment led to eventual loss of HIF1α(PP) expression, treated osteosarcoma U-2 OS cells acquired tumorigenicity in the subcutaneous tissue. Furthermore, the prior treatment resulted in widespread invasion of malignant glioma U-87 MG cells in the mouse brain and sustained growth of U-118 MG glioma cells. The lasting effects of HIF-1α on malignant progression are specific because neither HIF2α(PP) nor β-galactosidase yielded similar effects. By contrast, transient expression of HIF1α(PP) in U-87 MG cells or constitutive expression of HIF1α(PP) but not HIF2α(PP) in a patient-derived glioma sphere culture inhibited tumor growth and spread. Our results indicate that intermittent induction of HIF-1α produces lasting effects on malignant progression even at its own expense.

Annexin A2 regulates angiogenesis and invasion phenotypes of malignant glioma.

We have established a pair of animal models (J3T-1 and J3T-2) with different invasive and angiogenic phenotypes, and demonstrated that annexin A2 is expressed at higher levels in J3T-1 than J3T-2 cells. The function of annexin A2 in relation to angiogenesis and invasion was investigated using these models. Stable silencing or overexpression of annexin A2 in J3T-1 and J3T-2 cells (J3T-1shA and J3T-2A cells) was established and used. Thirty human glioblastoma samples were evaluated for expression of annexin A2, vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). Immunohistochemical and quantitative reverse-transcription polymerase chain reaction analyses revealed higher expression of annexin A2, VEGF and PDGF in J3T-1 and J3T-2A cells. Cultured J3T-1 and J3T-2A cells exhibited higher adhesive ability to endothelial cells. Histopathological analysis of animal brain tumors revealed that J3T-1 and J3T-2A tumors displayed marked angiogenesis and invasion along the neovasculature, whereas J3T-2 and J3T-1shA tumors exhibited diffuse, infiltrative invasion without angiogenesis. Positive expression of annexin A2 was observed in tumor cells surrounding dilated vessels in 25/30 human glioblastoma specimens. Our results reveal that the phenotype of glioma invasion is closely related to angiogenesis. We identify annexin A2 as a factor regulating angiogenesis and invasion of malignant gliomas.

ME-11 * REDUCTION OF NFkB IN THE IN THE TUMOR MICROENVIRONMENT ALTERS THE NEURAL EXTRACELLULAR MATRIX AND REDUCES GLIOBLASTOMA GROWTH AND DISPERSION

Identifying major support mechanisms in the neural microenvironment could have significant impact against growth and invasion of malignant gliomas. The NFkB pathway is a major signaling hub that drives tumor progression and at the same time regulates the inflammatory responses of normal tissues. Here we show that animals with a stunted NFkB response exhibit reduced reactivity against intracranial gliomas, which surprisingly reduces tumor growth and invasion. NFkB-deficient mice (RelA + /- TNFR1-/-) were injected intracranially with mouse GL261 glioblastoma cells to analyze tumor phenotype. Histological and MRI analyses showed significantly smaller tumors in RelA + /- mice compared to control littermates. Interestingly, immunohistochemical studies indicated that tumors in RelA + /- mice had vascularization and infiltration of microglia/macrophages comparable to controls, but significant reduction of M2 microglia/macrophage polarization and GFAP-positive astroglia. The lower reactivity of RelA + /- astroglia was also evident following intracranial implantation of highly invasive mouse glioblastoma cells (Mut4; GFAP-Cre:Nf1loxP/+ Trp53-/+ PTENloxP/+), suggesting that the astrocyte compartment played a major role in tumor growth or invasion. To test this hypothesis we prepared cultures of brain slices, where astrocytes and their secreted ECM are the major components challenging glioma cells. Interestingly, glioblastoma cells were significantly less invasive in brain slices from NFkB-deficient mice compared to wild-type tissue. Further analysis of RelA-/–astrocytes demonstrated reduced migration towards glioblastoma cells, increased expression of neural chondroitin sulfate proteoglycans and reduced expression of the metalloproteases that cleave these inhibitory ECM proteins, lending support to the reduced tumor progression observed in vivo. Taken together, these results strongly suggest that activation of NFkB in the neural microenvironment underlies the reactive glial response that promotes tumor growth and invasion. In particular, our results highlight astroglial NFkB signaling as a driver of glioma malignancy that should be considered for long-term targeted strategies.

Bradykinin enhances invasion of malignant glioma into the brain parenchyma by inducing cells to undergo amoeboid migration.

The molecular and cellular mechanisms governing cell motility and directed migration in response to the neuropeptide bradykinin are largely unknown. Here, we demonstrate that human glioma cells whose migration is guided by bradykinin generate bleb-like protrusions. We found that activation of the B2 receptor leads to a rise in free Ca(2+) from internal stores that activates actomyosin contraction and subsequent cytoplasmic flow into protrusions forming membrane blebs. Furthermore Ca(2+) activates Ca(2+)-dependent K(+) and Cl(-) channels, which participate in bleb regulation. Treatment of gliomas with bradykinin in situ increased glioma growth by increasing the speed of cell migration at the periphery of the tumour mass. To test if bleb formation is related to bradykinin-promoted glioma invasion we blocked glioma migration with blebbistatin, a blocker of myosin kinase II, which is necessary for proper bleb retraction. Our findings suggest a pivotal role of bradykinin during glioma invasion by stimulating amoeboid migration of glioma cells.

Abstract 4864: Tumor promoting role of NF-kappa B in the glioma environment

Abstract Introduction: Gliomas are the most common primary malignant brain tumors. Their characteristic features are extensive growth and lack of the clear borders due to diffuse invasion of surrounding tissue. Invasive glioma cells have been shown to interact through soluble signals with other cells in the neural microenvironment, which is normally highly inhibitory to cell motility. This communication changes the behavior of the tumor microenvironment to become permissive and facilitate tumor invasion. Here, we investigated the role of NFκB signaling in the glioma microenvironment and demonstrate that activation of NFκB in neural cells is critical for tumor growth and invasion. Methodology: We analyzed the growth and invasion of malignant gliomas using an NFκB-deficient mouse model (RelA(p65)-/- TNFR1-/-). Since p65-/- mice do not survive to adulthood, mouse glioma cells were implanted in p65+/- mice (which have significantly reduced NFκB activity) or their control littermates. Organotypic cultures of p65-/- brain slices and cultures of p65-/- neonatal astrocytes were used to assess the role of active NFκB signaling in astroglia on tumor invasion. Results: Using the mouse glioma cell line GL261 (poorly invasive) we observed significantly reduced tumor volume in NFκB -deficient mice compared to wild-type littermates. Further studies using mouse glioma stem-like cells Mut4 (highly invasive; GFAP-Cre: Nf1loxP/+ Trp53-/+ PTENloxP/+; Alcantara-Llaguno et al., Cancer Cell, 2009) demonstrated reduction both in tumor size and extent of invasion in the NFκB-deficient microenvironment. Importantly, immunohistochemical analysis demonstrated significantly reduced number of protoplasmic GFAP- positive astrocytes surrounding the tumors in p65+/- brains. Further analysis in brain slice cultures showed that invasion of glioblastoma cells was highly reduced through p65+/- tissue. Moreover, p65+/- astrocytes exhibited significantly reduced responses towards glioma cells, including reduced invasion through Matrigel in response to signals released by GL261 cells. Conclusions: Our results strongly suggest that NFκB signaling may be involved in the recruitment or activation of reactive astrocytes surrounding the invasive borders of gliomas. This activation is likely necessary for enhanced tumor invasion through brain tissue. Our results highlight the relevance of the astroglial compartment as a collateral target that should be considered in future strategies targeting brain tumor invasion. Citation Format: Aneta Kwiatkowska, Mohan Sobana Nandhu, Mariano Sebastian Viapiano. Tumor promoting role of NF-kappa B in the glioma environment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4864. doi:10.1158/1538-7445.AM2014-4864

P17.40 * GLIOMA ASSOCIATED MICROGLIAL MMP9 EXPRESSION IS UP REGULATED BY TLR2 SIGNALLING AND SENSITIVE TO MINOCYCLINE

OBJECTIVE: The invasiveness of malignant gliomas is one of the major obstacles in glioma therapy and the reason for the poor survival of patients. Glioma cells infiltrate into the brain parenchyma and thereby escape surgical resection. Glioma—associated microglia/macrophages (GAMs) support glioma infiltration into the brain parenchyma by increased expression and activation of extracellular matrix degrading proteases such as maxtrix—metalloprotease (MMP)—2, MMP—9 and MT1—MMP. METHODS: Using in vitro, ex vivo, and in vivo techniques, we identified TLR2 as the main TLR controlling microglial MMP9 expression and promoting microglia assisted glioma expansion. RESULTS: In this work we demonstrate that MMP—9 is predominantly expressed by GAMs in mouse and human glioma tissue but not by the glioma cells. Supernatant from glioma cells induced the expression of MMP—9 in cultured microglial cells. Using mice deficient for different toll—like receptors (TLRs) we identified TLR2/6 as the signalling pathway for the glioma induced upregulation of microglial MMP—9. Also in an experimental mouse glioma model, TLR2 deficiency attenuated the upregulation of microglial MMP—9. Moreover, glioma supernatant triggered an upregulation of TLR2 expression in microglia. Both, the upregulation of MMP—9 and TLR2 were attenuated by the antibiotic minocycline and a p38 MAPK antagonist in vitro. Minocycline also extended the survival rate of glioma bearing mice when given to the drinking water. CONCLUSIONS: Thus glioma cells change the phenotype of GAMs in a complex fashion using TLR2 as an important signalling pathway and minocycline further proved to be a potential candidate for adjuvant glioma therapy.

S1.02 * SUBLETHAL IRRADIATION ENHANCES INVASIVENESS OF MALIGNANT GLIOMA CELLS; IMPLICATIONS FOR RADIOTHERAPY OF HUMAN GLIOBLASTOMA

PURPOSE: This study explored the effect of irradiation on glioma invasion and also established relation between the infiltrative phenotype of glioma and the expression of p53 and MMP-2. METHODS: Human glioma cell line, U87MG (p53+) was prepared. U2OS (p53+) and SAOS2 (p53-) from osteosarcoma were used as control for p53 positive or negative. Each cell lines received single dose (2-8Gy) of irradiation, and then were analyzed the expression of p53, TIMP-1, 2 by western blot and MMP-2 activity by zymography. In order to confirm p53-MMP2 association, RNAi of p53 was treated into U87MG. The effect of irradiation on malignant glioma invasion was investigated by bioluminescence imaging after implantation U87-Fluc cell line in nude mouse brain, and then immunohistochemistry for p53, MMP-2 and in situ zymography were performed. RESULTS: MMP-2 activity and p53 expression were increased in proportional to irradiation dose in U87-MG and U2OS. TIMP-1, 2 was not changed in u87MG, but was increased in U2OS. In SAOS, TIMP-1, not TIMP-2 was increased after irradiation. Interestingly, MMP-2 began to express in 8 Gy-induced SAOS cells and expression of p53 and TIMP-1 were simultaneously increased. In vivo experiment, tumor margin of irradiated group was changed infiltrative phenotype, while control group was clear. MMP-2 and p53 was highly expressed in marginal area. And also, p53 knockdown decrease MMP-2 activity in irradiation induced u87MG cells. CONCLUSION: Irradiation could enhance glioma invasion, p53 expression and MMP-2 activities. Consequently p53 may regulate MMP-2 activity and high level of MMP-2 activation may play important roles in alteration to invasive growth pattern of U87MG by irradiation. These data suggest that the current involved-field radiotherapy for malignant glioma may need to be reconsidered and that future trials should consider pharmacologic therapies inhibiting invasion concurrently with radiotherapy.

Expression of α-subunit of α-glucosidase II in adult mouse brain regions and selected organs.

α-Glucosidase II (GII), a resident of endoplasmic reticulum (ER) and an important enzyme in the folding of nascent glycoproteins, is heterodimeric, consisting of α (GIIα) and β (GIIβ) subunits. The catalytic GIIα subunit, with the help of mannose 6-phosphate receptor homology domain of GIIβ, sequentially hydrolyzes two α1-3-linked glucose residues in the second step of N-linked oligosaccharide-mediated protein folding. The soluble GIIα subunit is retained in the ER through its interaction with the HDEL-containing GIIβ subunit. N-glycosylation and correct protein folding are crucial for protein stability and trafficking and cell surface expression of several proteins in the brain. Alterations in N-glycosylation lead to abnormalities in neuronal migration and mental retardation, various neurodegenerative diseases, and invasion of malignant gliomas. Inhibitors of GII are used to inhibit cell proliferation and migration in a variety of different pathologies, such as viral infection, cancer, and diabetes. Despite the widespread use of GIIα inhibitory drugs and the role of GIIα in brain function, little is known about its expression in brain and other tissues. Here, we report generation of a highly specific chicken antibody to the GIIα subunit and its characterization by Western blotting and immunoprecipitation using cerebral cortical extracts. By using this antibody, we showed that the GIIα protein is highly expressed in testis, kidney, and lung, with the lowest amount in heart. GIIα polypeptide levels in whole brain were comparable to those in spleen. However, a higher expression of GIIα protein was detected in the cerebral cortex, reflecting its continuous requirement in correct folding of cell surface proteins.

Glioma-associated microglial MMP9 expression is upregulated by TLR2 signaling and sensitive to minocycline.

The invasiveness of malignant gliomas is one of the major obstacles in glioma therapy and the reason for the poor survival of patients. Glioma cells infiltrate into the brain parenchyma and thereby escape surgical resection. Glioma associated microglia/macrophages support glioma infiltration into the brain parenchyma by increased expression and activation of extracellular matrix degrading proteases such as matrix metalloprotease (MMP) 2, MMP9 and membrane-type 1 MMP. In this work we demonstrate that, MMP9 is predominantly expressed by glioma associated microglia/macrophages in mouse and human glioma tissue but not by the glioma cells. Supernatant from glioma cells induced the expression of MMP9 in cultured microglial cells. Using mice deficient for different Toll-like receptors we identified Toll-like receptor 2/6 as the signaling pathway for the glioma induced upregulation of microglial MMP9. Also in an experimental mouse glioma model, Toll-like receptor 2 deficiency attenuated the upregulation of microglial MMP9. Moreover, glioma supernatant triggered an upregulation of Toll-like receptor 2 expression in microglia. Both, the upregulation of MMP9 and Toll-like receptor 2 were attenuated by the antibiotic minocycline and a p38 mitogen-activated protein kinase antagonist in vitro. Minocycline also extended the survival rate of glioma bearing mice when given to the drinking water. Thus glioma cells change the phenotype of glioma associated microglia/macrophages in a complex fashion using Toll-like receptor 2 as an important signaling pathway and minocycline further proved to be a potential candidate for adjuvant glioma therapy.