The NF-κB RelB Protein Is an Oncogenic Driver of Mesenchymal Glioma

High-grade gliomas, such as glioblastomas (GBMs), are very aggressive, invasive brain tumors with low patient survival rates. The recent identification of distinct glioma tumor subtypes offers the potential for understanding disease pathogenesis, responses to treatment and identification of molecular targets for personalized cancer therapies. However, the key alterations that drive tumorigenesis within each subtype are still poorly understood. Although aberrant NF-κB activity has been implicated in glioma, the roles of specific members of this protein family in tumorigenesis and pathogenesis have not been elucidated. In this study, we show that the NF-κB protein RelB is expressed in a particularly aggressive mesenchymal subtype of glioma, and loss of RelB significantly attenuated glioma cell survival, motility and invasion. We find that RelB promotes the expression of mesenchymal genes including YKL-40, a marker of the MES glioma subtype. Additionally, RelB regulates expression of Olig2, a regulator of cancer stem cell proliferation and a candidate marker for the cell of origin in glioma. Furthermore, loss of RelB in glioma cells significantly diminished tumor growth in orthotopic mouse xenografts. The relevance of our studies for human disease was confirmed by analysis of a human GBM genome database, which revealed that high RelB expression strongly correlates with rapid tumor progression and poor patient survival rates. Thus, our findings demonstrate that RelB is an oncogenic driver of mesenchymal glioma tumor growth and invasion, highlighting the therapeutic potential of inhibiting the noncanonical NF-κB (RelB-mediated) pathway to treat these deadly tumors.     

Toward a molecular classification of the gliomas: histopathology, molecular genetics, and gene expression profiling

As many as 100,000 new cases of brain tumor are diagnosed each year in the United States. About half of these are primary gliomas and the remaining half are metastatic tumors and non-glial primary tumors. Currently, gliomas are classified based on phenotypic characteristics. Recent progress in the elucidation of genetic alterations found in gliomas have raised the exciting possibility of using genetic and molecular analyses to resolve some of the problematic issues currently associated with the histological approach to glioma classification. Recently, immunohistochemical studies using novel proliferation markers have significantly advanced the assessment of tumor growth potential and the grading criteria of some tumor subtypes. Preliminary studies using cDNA array technologies suggest that the profiling of gene expression patterns may provide a novel and meaningful approach to glioma classification and subclassification. Furthermore, cDNA array technologies may also be used to identify candidate genes involved in glioma tumor development, invasion, and progression. This review summarizes current glioma classification schemes that are based on histopathological characteristics and discusses the potential for using cDNA array technology in the molecular classification of gliomas.     

The Different Role of Notch1 and Notch2 in Astrocytic Gliomas

It is well known that Notch signaling plays either oncogenic or tumor suppressive role in a variety of tumors, depending on the cellular context. However, in our previous study, we found that Notch1 was overexpressed while Notch2 downregulated in the majority of astrocytic gliomas with different grades as well as in glioblastoma cell lines U251 and A172. We had knocked down Notch1 by siRNA in glioblastoma cells, and identified that the cell growth and invasion were inhibited, whereas cell apoptosis was induced either in vitro or in vivo. For further clarification of the role of Notch2 in pathogenesis of gliomas, enforced overexpression of Notch2 was carried out with transfection of Notch2 expression plasmid in glioma cells and the cell growth, invasion and apoptosis were examined in vitro and in vivo in the present study, and siRNA targeting Notch1 was used as a positive control in vivo. The results showed that upregulating Notch2 had the effect of suppressing cell growth and invasion as well as inducing apoptosis, just the same as the results of knocking down Notch1. Meanwhile, the activity of core signaling pathway–EGFR/PI3K/AKT in astrocytic glioma cells was repressed. Thus, the present study reveals, for the first time, that Notch1 and Notch2 play different roles in the biological processes of astrocytic gliomas. Knocking down the Notch1 or enforced overexpression of Notch2 both modulate the astrocytic glioma phenotype, and the mechanism by which Notch1 and 2 play different roles in the glioma growth should be further investigated.     

The Guanine Nucleotide Exchange Factor SWAP-70 Modulates the Migration and Invasiveness of Human Malignant Glioma Cells

The malignant glioma is the most common primary human brain tumor. Its tendency to invade away from the primary tumor mass is considered a leading cause of tumor recurrence and treatment failure. Accordingly, the molecular pathogenesis of glioma invasion is currently under investigation. Previously, we examined a gene expression array database comparing human gliomas to nonneoplastic controls and identified several Rac guanine nucleotide exchange factors with differential expression. Here, we report that the guanine nucleotide exchange factor SWAP-70 has increased expression in malignant gliomas and strongly correlates with lowered patient survival. SWAP-70 is a multifunctional signaling protein involved in membrane ruffling that works cooperatively with activated Rac. Using a glioma tissue microarray, we validated that SWAP-70 demonstrates higher expression in malignant gliomas compared with low-grade gliomas or nonneoplastic brain tissue. Through immunofluorescence, SWAP-70 localizes to membrane ruffles in response to the growth factor, epidermal growth factor. To assess the role of SWAP-70 in glioma migration and invasion, we inhibited its expression withsmall interfering RNAs and observed decreased glioma cell migration and invasion. SWAP-70 overexpression led to increased levels of active Rac even in low-serum conditions. In addition, when SWAP-70 was overexpressed in glioma cells, we observed enhanced membrane ruffle formation followed by increased cellmigration and invasiveness. Taken together, our findings suggest that the guanine nucleotide exchange factor SWAP-70 plays an important role in the migration and invasion of human gliomas into the surrounding tissue.     

Potential adenovirus-mediated gene therapy of glioma cancer

Malignant gliomas are typically characterized by rapid cell proliferation and a marked propensity to invade and damage surrounding tissues. They are the main brain tumors notoriously resistant to currently available therapies, since they fail to undergo apoptosis upon anticancer treatments. With recent advances in neuroscience and improved understanding of the molecular mechanisms of invasive migration, gene therapy provides a new strategy for treating glioma cancer. Brain tumor gene therapy using viral vectors and stem cells has shown promise in animal model and human patient studies. Here, we review recent studies on engineering adenoviral vectors that can be used as therapy for brain tumors. The new findings presented in this study are essential for the further exploration of this cancer and they represent an approach for developing a newer and more effective therapeutic approach in the clinical treatment of human glioma cancer.     

Downregulation of RKIP is associated with poor outcome and malignant progression in gliomas

Malignant gliomas are highly infiltrative and invasive tumors, which precludes the few treatment options available. Therefore, there is an urgent need to elucidate the molecular mechanisms underlying gliomas aggressive phenotype and poor prognosis. The Raf Kinase Inhibitory protein (RKIP), besides regulating important intracellular signaling cascades, was described to be associated with progression, metastasis and prognosis in several human neoplasms. Its role in the prognosis and tumourigenesis of gliomas remains unclear. In the present study, we found that RKIP protein is absent in a low frequency (10%, 20/193) of glioma tumors. Nevertheless, the absence of RKIP expression was an independent prognostic marker in glioma. Additionally, by in vitro downregulation of RKIP, we found that RKIP inhibition induces a higher viability and migration of the cells, having no effect on cellular proliferation and angiogenesis, as assessed by in vivo CAM assay. In conclusion, this is the largest series studied so far evaluating the expression levels of this important cancer suppressor protein in glioma tumors. Our results suggest that in a subset of tumors, the absence of RKIP associates with highly malignant behavior and poor survival of patients, which may be a useful biomarker for tailored treatment of glioma patients.     

Role of NOD- like Receptors in Glioma Angiogenesis: Insights into future therapeutic interventions

Gliomas are the most common solid tumors among central nervous system tumors. Most glioma patients succumb to their disease within two years of the initial diagnosis. The median survival of gliomas is only 14.6 months, even after aggressive therapy with surgery, radiation, and chemotherapy. Gliomas are heavily infiltrated with myeloid- derived cells and endothelial cells. Increasing evidence suggests that these myeloid- derived cells interact with tumor cells promoting their growth and migration. NLRs (nucleotide-binding oligomerization domain (NOD)-containing protein like receptors) are a class of pattern recognition receptors that are critical to sensing pathogen and danger associated molecular patterns. Mutations in some NLRs lead to autoinflammatory diseases in humans. Moreover, dysregulated NLR signaling is central to the pathogenesis of several cancers, autoimmune and neurodegenerative diseases. Our review explores the role of angiogenic factors that contribute to upstream or downstream signaling pathways leading to NLRs. Angiogenesis plays a significant role in the pathogenesis of variety of tumors including gliomas. Though NLRs have been detected in several cancers including gliomas and NLR signaling contributes to angiogenesis, the exact role and mechanism of involvement of NLRs in glioma angiogenesis remain largely unexplored. We discuss cellular, molecular and genetic studies of NLR signaling and convergence of NLR signaling pathways with angiogenesis signaling in gliomas. This may lead to re-appropriation of existing anti-angiogenic therapies or development of future strategies for targeted therapeutics in gliomas.     

Genetic and epigenetic markers of gliomas

Malignant gliomas are aggressive and highly invasive tumors. Various genetic and epigenetic changes are common for these tumors. Mostly they concern the genes involved in cell-cycle regulation, apoptotic pathways, cell invasion, angiogenesis, and cell metabolism. The role of epigenetic mechanisms in glioma malignant transformation, despite recent progress, is uncertain and remains under intense study. This review describes the mechanisms of epigenetic regulation of gene expression, including posttranslational modifications of histones, DNA methylation in promoter regions, and microRNA regulation. The genetic and epigenetic factors driving the pathogenesis of gliomas in their possible mutual influence and the potential epigenetic targets that can be used for diagnostics and new therapeutic approaches are also discussed.     

The emerging role of MMP14 in brain tumorigenesis and future therapeutics

Glioblastoma is a malignant brain tumor of glial origin. These tumors are thought to be derived from astrocytic cells that undergo malignant transformation. A growing body of evidence suggests that upregulation of MMP expression plays a significant role in promoting glioma pathogenesis. Elevated expression of MMP14 not only promotes glioma invasion and tumor cell proliferation but also plays a role in angiogenesis. Despite the fact that levels of MMP14 correlate with breast cancer progression, the controversial role of MMP14 in gliomagenesis needs to be elucidated. In the present review, we discuss the role of MMP14 in glioma progression as well as the mechanisms of MMP14 regulation in the context of future therapeutic manipulations.     

Direct inhibition of myosin II effectively blocks glioma invasion in the presence of multiple motogens

Anaplastic gliomas, the most common and malignant of primary brain tumors, frequently contain activating mutations and amplifications in promigratory signal transduction pathways. However, targeting these pathways with individual signal transduction inhibitors does not appreciably reduce tumor invasion, because these pathways are redundant; blockade of any one pathway can be overcome by stimulation of another. This implies that a more effective approach would be to target a component at which these pathways converge. In this study, we have investigated whether the molecular motor myosin II represents such a target by examining glioma invasion in a series of increasingly complex models that are sensitive to platelet-derived growth factor, epidermal growth factor, or both. Our results lead to two conclusions. First, malignant glioma cells are stimulated to invade brain through the activation of multiple signaling cascades not accounted for in simple in vitro assays. Second, even though there is a high degree of redundancy in promigratory signaling cascades in gliomas, blocking tumor invasion by directly targeting myosin II remains effective. Our results thus support our hypothesis that myosin II represents a point of convergence for signal transduction pathways that drive glioma invasion and that its inhibition cannot be overcome by other motility mechanisms.     

Epigenetic Aberrations in Malignant Gliomas: An Open Door Leading to Better Understanding and Treatment

Malignant gliomas and specially glioblastoma multiforme are the most frequent and devastating brain tumors in adults. Intensive molecular and cytogenetical studies have revealed a wide variety of deregulated genes implicated in cell cyclus, DNA repair, apoptosis, cell migration, invasion and angiogenesis with little translational success. An increasing number of reports investigating epigenetic injuries in malignant gliomas have been recently published, although the panorama of CpG island aberrant hypermethylation, histone modification and chromatin states in these lethal tumors is only partially devised. In the present analysis, we discuss the magnitude and significance of epigenetic lesions in the pathogenesis and mechanisms of progression of malignant gliomas as well as their influence on patient survival. The new venue of epigenetic research provides tools for the identification of genes differentially methylated that may be implicated in tumorigenesis and furthermore, epigenetics-based drugs may constitute a promising alternative resource of therapy for this, to the moment, incurable malignancy.     

The emerging role of miR‐19 in glioma

Glioma has been regarded as the most common, highly proliferative and invasive brain tumour. Advances in research of miRNAs in glioma are toward further understanding of the pathogenesis of glioma. MiR‐19, a member of miR‐17~92 cluster, was reported to play an oncogenic role in tumourigenesis. Here we review the identified data about the effect of miR‐19 on proliferation, apoptosis, migration and invasion of glioma cells, the target genes regulated by miR‐19, and correlation of miR‐19 with the sensitivity of glioma cells to chemotherapy and radiotherapy. It is concluded that miR‐19 plays an important role in the pathogenesis of glioma and can be a potential target for gene therapy of glioma.     

microRNA-34a is tumor suppressive in brain tumors and glioma stem cells

We recently found that microRNA-34a (miR-34a) is downregulated in human glioma tumors as compared to normal brain, and that miR-34a levels in mutant-p53 gliomas were lower than in wildtype-p53 tumors. We showed that miR-34a expression in glioma and medulloblastoma cells inhibits cell proliferation, G1/S cell cycle progression, cell survival, cell migration and cell invasion, but that miR-34a expression in human astrocytes does not affect cell survival and cell cycle. We uncovered the oncogenes c-Met, Notch-1 and Notch-2 as direct targets of miR-34a that are inhibited by miR-34a transfection. We found that c-Met levels in human glioma specimens inversely correlate with miR-34a levels. We showed that c-Met and Notch partially mediate the inhibitory effects of miR-34a on cell proliferation and cell death. We also found that mir-34a expression inhibits in vivo glioma xenograft growth. We concluded that miR-34a is a potential tumor suppressor in brain tumors that acts by targeting multiple oncogenes. In this extra view, we briefly review and discuss the implications of these findings and present new data on the effects of miR-34a in glioma stem cells. The new data show that miR-34a expression inhibits various malignancy endpoints in glioma stem cells. Importantly, they also show for the first time that miR-34a expression induces glioma stem cell differentiation. Altogether, the data suggest that miR-34a is a tumor suppressor and a potential potent therapeutic agent that acts by targeting multiple oncogenic pathways in brain tumors and by inducing the differentiation of cancer stem cells.     

Interferon gamma inhibits proliferation and hyaluronic acid adhesion of human malignant glioma cells in vitro

Malignant gliomas are frequent and the prognosis is poor. The cytokine interferon gamma (IFN-gamma) enhances several immune phenomena and may be used in immunotherapy of tumours. Therefore we investigated the influence of IFN-gamma on human cell lines T98G, U87MG, 86HG39 and 85HG66, measuring cell viability (MTT-test) and proliferation (3H-thymidine uptake). IFN-gamma markedly decreased viability and proliferation of all investigated cell lines. Expression of CD44 and adhesion to hyaluronic acid (HA) are involved in glioma invasion. Influence of IFN-gamma on these two features has also been investigated. IFN-gamma markedly decreased HA-adhesion in all three investigated cell lines, whereas CD44 expression remained uninfluenced. To summarise, IFN-gamma strongly decreased cell growth and HA-adhesion of malignant glioma cell lines in vitro. We suggest further investigations to characterise better the role of IFN-gamma as a treatment opportunity for malignant gliomas.     

The pro-migratory and pro-invasive role of the procoagulant tissue factor in malignant gliomas

During the infiltration process, glioma cells are known to migrate along preexisting anatomical structures such as blood vessels, axonal fiber tracts and the subependymal space, thereby widely invading surrounding CNS tissue. This phenomenon represents a major obstacle for the clinical treatment of these tumours. Several extracellular key factors and intracellular signaling pathways have been previously linked to the highly aggressive, invasive phenotype observed in malignant gliomas. The glioblastoma (GBM) which is the most malignant form of these tumors, is histologically characterized by areas of tumor necroses and pseudopalisading cells, the latter likely representing tumor cells actively migrating away from the hypoxic-ischemic core of the tumor. It is believed that intravascular thromboses play a major role in the emergence of hypoxia and intratumoral necroses in GBMs. One of the most highly upregulated prothrombotic factor in malignant gliomas is tissue factor (TF), a 47 kDa type I transmembrane protein belonging to the cytokine receptor superfamily. In a recent study, we provided evidence that TF/FVIIa signaling via the protease-activated receptor 2 (PAR-2) promotes cell growth, migration and invasion of glioma cells. In this point of view article we outline the key molecular players involved in migration and invasion of gliomas, highlight the potential role of TF for the pro-migratory and pro-invasive phenotype of these tumors and discuss the underlying mechanisms on the cellular level and in the tumor microenvironment.     

Role of hyaluronan in glioma invasion

Gliomas are the most common primary intracranial tumors. Their distinct ability to infiltrate into the extracellular matrix (ECM) of the brain makes it impossible to treat these tumors using surgery and radiation therapy. A number of different studies have suggested that hyaluronan (HA), the principal glycosaminoglycan (GAG) in the ECM of the brain, is the critical factor for glioma invasion. HA-induced glioma invasion was driven by two important molecular events: matrix metalloproteinase (MMP) secretion and upregulation of cell migration. MMP secretion was triggered by HA-induced focal adhesion kinase (FAK) activation, which transmits its signal through ERK activation and nuclear factor kappa B (NFκB) translocation. Another important molecular event is osteopontin (OPN) expression. OPN expression by AKT activation triggers cell migration. These results suggest that HA-induced glioma invasion is tightly regulated by signaling mechanisms, and a detailed understanding of this molecular mechanism will provide important clues for glioma treatment.Key words: hyaluronan, matrix metalloproteinase, osteopontin, emodin, invasion, gliomaMalignant gliomas are highly invasive and infiltrative tumors that have a poor prognosis with a median survival of only one year.^1,2 A major barrier to effective malignant glioma treatment is the invasion of these cells into brain parenchyma. Because of this fact, local therapies such as surgery or radiation therapy are not effective.³ Glioma cells invade through the ECM of the brain by activating a number of coordinated cellular programs, which include those necessary for migration and invasion.³ Therefore, a detailed understanding of the mechanisms underlying this invasive behavior is essential for the development of novel effective therapies.During glioma invasion, tumor cells closely interact with the ECM. Although brain tumor cells may share some of the invasive characteristics with tumors that arise outside of the central nerve system (CNS), the particular structure and composition of the brain ECM suggest the existence of unique invasive mechanisms for brain tumors.⁴Brain ECM is composed of typical ECM proteins and a HA scaffold with associated glycoproteins and proteoglycans.⁵ Typical ECM proteins such as laminin, type-IV collagen and fibronectin have been implicated in the invasion of other tumors by regulating cell adhesion and migration.⁶ However HA, which is associated with proteoglycans and GAGs, is especially abundant in the brain parenchyma compared to other tissues.⁷ Furthermore, malignant gliomas contain higher amounts of HA than normal brain tissue.⁷ These facts raise the possibility that HA might play an important role in glioma invasion, a process that is distinct from other non-CNS derived tumors.     

The role of PKN1 in glioma pathogenesis and the antiglioma effect of raloxifene targeting PKN1

PKN1 (protein kinase N1), a serine/threonine protein kinase family member, is associated with various cancers. However, the role of PKN1 in gliomas has rarely been studied. We suggest that PKN1 expression in glioma specimens is considerably upregulated and positively correlates with the histopathological grading of gliomas. Knocking down PKN1 expression in glioblastoma (GBM) cells inhibits GBM cell proliferation, invasion and migration and promotes apoptosis. In addition, yes-associated protein (YAP) expression, an essential effector of the Hippo pathway contributing to the oncogenic role of gliomagenesis, was also downregulated. In contrast, PKN1 upregulation enhances the malignant characteristics of GBM cells and simultaneously upregulates YAP expression. Therefore, PKN1 is a promising therapeutic target for gliomas. Raloxifene (Ralo), a commonly used selective oestrogen-receptor modulator to treat osteoporosis in postmenopausal women, was predicted to target PKN1 according to the bioinformatics team from the School of Mathematics, Tianjin Nankai University. We showed that Ralo effectively targets PKN1, inhibits GBM cells proliferation and migration and sensitizes GBM cells to the major chemotherapeutic drug, Temozolomide. Ralo also reverses the effect of PKN1 on YAP activation. Thus, we confirm that PKN1 contributes to the pathogenesis of gliomas and may be a potential target for Ralo adjuvant glioma therapy.