HEDGEHOG-GLI1 signaling regulates human glioma growth, cancer stem cell self-renewal, and tumorigenicity

Cancer stem cells are rare tumor cells characterized by their ability to self-renew and to induce tumorigenesis. They are present in gliomas and may be responsible for the lethality of these incurable brain tumors. In the most aggressive and invasive type, glioblastoma multiforme (GBM), an average of about one year spans the period between detection and death [1]. The resistence of gliomas to current therapies may be related to the existence of cancer stem cells [2-6]. We find that human gliomas display a stemness signature and demonstrate that HEDGEHOG (HH)-GLI signaling regulates the expression of stemness genes in and the self-renewal of CD133(+) glioma cancer stem cells. HH-GLI signaling is also required for sustained glioma growth and survival. It displays additive and synergistic effects with temozolomide (TMZ), the current chemotherapeutic agent of choice. TMZ, however, does not block glioma stem cell self-renewal. Finally, interference of HH-GLI signaling with cyclopamine or through lentiviral-mediated silencing demonstrates that the tumorigenicity of human gliomas in mice requires an active pathway. Our results reveal the essential role of HH-GLI signaling in controlling the behavior of human glioma cancer stem cells and offer new therapeutic possibilities.     

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.     

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.     

Proteomics-based identification of differentially expressed genes in human gliomas: down-regulation of SIRT2 gene

A number of chromosomal abnormalities including 19q deletions have been associated with the formation of human gliomas. In this study, we employed a proteomics-based approach to identify possible genes involved in glioma tumorigenesis which may serve as potential diagnostic molecular markers for this type of cancer. By comparing protein spots from gliomas and non-tumor tissues using two-dimensional (2D) gel electrophoresis, we identified 11 up-regulated proteins and four down-regulated proteins in gliomas. Interestingly, we also discovered that a group of cytoskeleton-related proteins are differentially regulated in gliomas, suggesting the involvement of cytoskeleton modulation in glioma pathogenesis. We then focused on the cytoskeleton-related protein, SIRT2 (sirtuin homologue 2) tubulin deacetylase, which was down-regulated in gliomas. SIRT2 is located at 19q13.2, a region known to be frequently deleted in human gliomas. Subsequent Northern blot analysis revealed that RNA expression of SIRT2 was dramatically diminished in 12 out of 17 gliomas and glioma cell lines, in agreement with proteomic data. Furthermore, ectopic expression of SIRT2 in glioma cell lines led to the perturbation of the microtubule network and caused a remarkable reduction in the number of stable clones expressing SIRT2 as compared to that of a control vector in colony formation assays. These results suggest that SIRT2 may act as a tumor suppressor gene in human gliomas possibly through the regulation of microtubule network and may serve as a novel molecular marker for gliomas. Additional proteins were also identified, whose function in gliomas was previously unsuspected.     

Stimulation of the midkine/ALK axis renders glioma cells resistant to cannabinoid antitumoral action

Identifying the molecular mechanisms responsible for the resistance of gliomas to anticancer treatments is an issue of great therapeutic interest. Δ(9)-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoids inhibit tumor growth in animal models of cancer, including glioma, an effect that relies, at least in part, on the stimulation of autophagy-mediated apoptosis in tumor cells. Here, by analyzing the gene expression profile of a large series of human glioma cells with different sensitivity to cannabinoid action, we have identified a subset of genes specifically associated to THC resistance. One of these genes, namely that encoding the growth factor midkine (Mdk), is directly involved in the resistance of glioma cells to cannabinoid treatment. We also show that Mdk mediates its protective effect via the anaplastic lymphoma kinase (ALK) receptor and that Mdk signaling through ALK interferes with cannabinoid-induced autophagic cell death. Furthermore, in vivo Mdk silencing or ALK pharmacological inhibition sensitizes cannabinod-resistant tumors to THC antitumoral action. Altogether, our findings identify Mdk as a pivotal factor involved in the resistance of glioma cells to THC pro-autophagic and antitumoral action, and suggest that selective targeting of the Mdk/ALK axis could help to improve the efficacy of antitumoral therapies for gliomas.     

Sleeping Beauty Mouse Models Identify Candidate Genes Involved in Gliomagenesis

Genomic studies of human high-grade gliomas have discovered known and candidate tumor drivers. Studies in both cell culture and mouse models have complemented these approaches and have identified additional genes and processes important for gliomagenesis. Previously, we found that mobilization of Sleeping Beauty transposons in mice ubiquitously throughout the body from the Rosa26 locus led to gliomagenesis with low penetrance. Here we report the characterization of mice in which transposons are mobilized in the Glial Fibrillary Acidic Protein (GFAP) compartment. Glioma formation in these mice did not occur on an otherwise wild-type genetic background, but rare gliomas were observed when mobilization occurred in a p19Arf heterozygous background. Through cloning insertions from additional gliomas generated by transposon mobilization in the Rosa26 compartment, several candidate glioma genes were identified. Comparisons to genetic, epigenetic and mRNA expression data from human gliomas implicates several of these genes as tumor suppressor genes and oncogenes in human glioblastoma.     

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 mitochondrial genome is a "genetic sanctuary" during the oncogenic process

Since Otto Warburg linked mitochondrial physiology and oncogenesis in the 1930s, a number of studies have focused on the analysis of the genetic basis for the presence of aerobic glycolysis in cancer cells. However, little or no evidence exists today to indicate that mtDNA mutations are directly responsible for the initiation of tumor onset. Based on a model of gliomagenesis in the mouse, we aimed to explore whether or not mtDNA mutations are associated with the initiation of tumor formation, maintenance and aggressiveness. We reproduced the different molecular events that lead from tumor initiation to progression in the mouse glioma. In human gliomas, most of the genetic alterations that have been previously identified result in the aberrant activation of different signaling pathways and deregulation of the cell cycle. Our data indicates that mitochondrial dysfunction is associated with reactive oxygen species (ROS) generation, leading to increased nuclear DNA (nDNA) mutagenesis, but maintaining the integrity of the mitochondrial genome. In addition, mutational stability has been observed in entire mtDNA of human gliomas; this is in full agreement with the results obtained in the cancer mouse model. We use this model as a paradigm of oncogenic transformation due to the fact that mutations commonly found in gliomas appear to be the most common molecular alterations leading to tumor development in most types of human cancer. Our results indicate that the mtDNA genome is kept by the cell as a "genetic sanctuary" during tumor development in the mouse and humans. This is compatible with the hypothesis that the mtDNA molecule plays an essential role in the control of the cellular adaptive survival response to tumor-induced oxidative stress. The integrity of mtDNA seems to be a necessary element for responding to the increased ROS production associated with the oncogenic process.     

Recruited cells can become transformed and overtake PDGF-induced murine gliomas in vivo during tumor progression

Background

Gliomas are thought to form by clonal expansion from a single cell-of-origin, and progression-associated mutations to occur in its progeny cells. Glioma progression is associated with elevated growth factor signaling and loss of function of tumor suppressors Ink4a, Arf and Pten. Yet, gliomas are cellularly heterogeneous; they recruit and trap normal cells during infiltration.

Methodology/Principal Findings

We performed lineage tracing in a retrovirally mediated, molecularly and histologically accurate mouse model of hPDGFb-driven gliomagenesis. We were able to distinguish cells in the tumor that were derived from the cell-of-origin from those that were not. Phenotypic, tumorigenic and expression analyses were performed on both populations of these cells. Here we show that during progression of hPDGFb-induced murine gliomas, tumor suppressor loss can expand the recruited cell population not derived from the cell-of-origin within glioma microenvironment to dominate regions of the tumor, with essentially no contribution from the progeny of glioma cell-of-origin. Moreover, the recruited cells can give rise to gliomas upon transplantation and passaging, acquire polysomal expression profiles and genetic aberrations typically present in glioma cells rather than normal progenitors, aid progeny cells in glioma initiation upon transplantation, and become independent of PDGFR signaling.

Conclusions/Significance

These results indicate that non-cell-of-origin derived cells within glioma environment in the mouse can be corrupted to become bona fide tumor, and deviate from the generally established view of gliomagenesis.     

Molecular biology of glioma tumorigenesis

Gliomas are the most common intracranial malignant tumors in humans, and high-grade gliomas in particular pose a unique challenge due to their propensity for proliferation and tissue invasion. Our understanding of glioma oncogenesis, proliferation, and invasion has been greatly advanced in the past 10 years as researchers have gained a better understanding of the molecular biology of these tumors. This article highlights glioma histopathology, as well as cytogenetic and molecular alterations associated with the pathogenesis of human gliomas. It is hoped that better understanding of the molecular pathogenesis of gliomas will improve tumor classification as well as lead to novel targets for therapy and prognostic markers.     

TROY (TNFRSF19) is overexpressed in advanced glial tumors and promotes glioblastoma cell invasion via Pyk2-Rac1 signaling

A critical problem in the treatment of malignant gliomas is the extensive infiltration of individual tumor cells into adjacent brain tissues. This invasive phenotype severely limits all current therapies, and to date, no treatment is available to control the spread of this disease. Members of the tumor necrosis factor (TNF) ligand superfamily and their cognate receptors regulate various cellular responses including proliferation, migration, differentiation, and apoptosis. Specifically, the TNFRSF19/TROY gene encodes a type I cell surface receptor that is expressed on migrating or proliferating progenitor cells of the hippocampus, thalamus, and cerebral cortex. Here, we show that levels of TROY mRNA expression directly correlate with increasing glial tumor grade. Among malignant gliomas, TROY expression correlates inversely with overall patient survival. In addition, we show that TROY overexpression in glioma cells activates Rac1 signaling in a Pyk2-dependent manner to drive glioma cell invasion and migration. Pyk2 coimmunoprecipitates with the TROY receptor, and depletion of Pyk2 expression by short hairpin RNA interference oligonucleotides inhibits TROY-induced Rac1 activation and subsequent cellular migration. These findings position aberrant expression and/or signaling by TROY as a contributor, and possibly as a driver, of the malignant dispersion of glioma cells.     

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models. To model WHO grades III and IV malignant gliomas in transplantation assays, human tumor cells are xenografted into an orthotopic site, the brain, of immunocompromised mice. In contrast to secondary xenografts that utilize cultured tumor cells, human glioma cells are dissociated from resected specimens and transplanted without prior passage in tissue culture to generate primary xenografts. The procedure in this report details tumor sample preparation, intracranial transplantation into immunocompromised mice, monitoring for tumor engraftment and tumor harvesting for subsequent passage into recipient animals or analysis. Tumor cell preparation requires 2 hr and surgical procedure requires 20 min/animal.     

Autocrine BMP4 Signaling Enhances Tumor Aggressiveness via Promoting Wnt/β-Catenin Signaling in IDH1-mutant Gliomas

The isocitrate dehydrogenase (IDH1/2) mutations are frequent genetic abnormalities in the majority of WHO grade II/III glioma and secondary GBM. IDH1-mutated (IDH1^Mut) glioma exhibits distinctive patterns in cancer biology and metabolism. In the present study, we showed that bone morphogenetic proteins (BMP4) are significantly upregulated in IDH1^Mut glioma. Further, we demonstrated that cancer-associated BMP4 is secreted to tumor microenvironment, which enhances the tumor migration and invasion through an autocrine manner. Mechanistically, BMP4 activates its receptor and concomitant SMAD1/5/8 signaling, which potentiates Wnt/β-catenin signaling by enhancing Frizzled receptor expression. LDN-193189, a selective BMP receptor inhibitor, prolonged the overall survival of mice bearing IDH1-mutated intracranial xenografts by limiting BMP/catenin signaling. These findings demonstrate the pivotal role of BMP4 on tumor aggressiveness in IDH1^Mut gliomas, suggesting a possible therapeutic strategy for this type of malignancy.     

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.     

A role for the NG2 proteoglycan in glioma progression

Many human gliomas carry markers characteristic of oligodendrocyte progenitor cells (such as Olig-2, PDGF alpha receptor, and NG2 proteoglycan), suggesting these progenitors as the cells of origin for glioma initiation. This review considers the potential roles of the NG2 proteoglycan in glioma progression. NG2 is expressed not only by glioma cells and by oligodendrocyte progenitors, but also by pericytes associated with the tumor microvasculature. The proteoglycan may therefore promote tumor vascularization and recruitment of normal progenitors to the tumor mass, in addition to mediating expansion of the transformed cell population. Along with potentiating growth factor signaling and serving as a cell surface receptor for extracellular matrix components, NG2 also has the ability to mediate activation of beta-1 integrins. These molecular interactions allow the proteoglycan to contribute to critical processes such as cell proliferation, cell motility, and cell survival.