Global microRNA profiling identified miR-10b-5p as a regulator of neurofibromatosis 1 (NF1)-glioma migration.

AIMS: Neurofibromatosis 1 (NF1) is an autosomal-dominant cancer predisposition syndrome caused by loss of function alterations involving the NF1 locus on chromosome 17. The most common brain tumours encountered in affected patients are low-grade gliomas (pilocytic astrocytomas), although high-grade gliomas are also observed at increased frequency. While bi-allelic NF1 loss characterizes these tumours, previous studies have suggested noncoding RNA molecules (microRNA, miR) may have important roles in dictating glioma biology. METHODS: To explore the contributions of miRs in NF1-associated gliomas, we analysed five high-grade gliomas (NF1-HGG) and five PAs (NF1-PA) using global microRNA profiling with NanoString-based microarrays followed by functional experiments with glioma cell lines. RESULTS: miR-10b-5p, miR-135b-5p, miR-196a-5p, miR-196b-5p, miR-1247-5p and miR-320a (adjusted P < 0.05) were increased> 3-fold in NF1-HGG relative to NF1-PA tumours. In addition, miR-378b and miR-1305 were decreased 6.8- and 6-fold, respectively, whereas miR-451a was increased 2.7-fold (adjusted P < 0.05) in NF1-PAs compared to non-neoplastic NF1 patient brain specimens (n = 2). As miR-10b-5p was the microRNA overexpressed the most in NF1-high-grade glioma compared to NF1-low-grade glioma (5.76 fold), we examined its levels in glioma cell lines. miR-10b-5p levels were highest in adult glioma cell lines and lowest in paediatric low-grade glioma lines (P = 0.02). miR-10b-5p knockdown resulted in decreased invasion in NF1-deficient LN229 high-grade glioma line, whereas its overexpression in the NF1-PA derived line (JHH-NF1-PA1) led to increased invasion. There was no change in cell growth (viability and proliferation). CONCLUSIONS: These proof-of-concept experiments support a role for microRNA regulation in NF1-glioma biology.

HCN channels are a novel therapeutic target for cognitive dysfunction in Neurofibromatosis type 1.

Cognitive impairments are a major clinical feature of the common neurogenetic disease neurofibromatosis type 1 (NF1). Previous studies have demonstrated that increased neuronal inhibition underlies the learning deficits in NF1, however, the molecular mechanism underlying this cell-type specificity has remained unknown. Here, we identify an interneuron-specific attenuation of hyperpolarization-activated cyclic nucleotide-gated (HCN) current as the cause for increased inhibition in Nf1 mutants. Mechanistically, we demonstrate that HCN1 is a novel NF1-interacting protein for which loss of NF1 results in a concomitant increase of interneuron excitability. Furthermore, the HCN channel agonist lamotrigine rescued the electrophysiological and cognitive deficits in two independent Nf1 mouse models, thereby establishing the importance of HCN channel dysfunction in NF1. Together, our results provide detailed mechanistic insights into the pathophysiology of NF1-associated cognitive defects, and identify a novel target for clinical drug development.

The molecular and cell biology of pediatric low-grade gliomas.

Pilocytic astrocytoma (PA) is the most common glial cell tumor arising in children. Sporadic cases are associated with KIAA1549:BRAF fusion rearrangements, while 15-20% of children develop PA in the context of the neurofibromatosis 1 (NF1) inherited tumor predisposition syndrome. The unique predilection of these tumors to form within the optic pathway and brainstem (NF1-PA) and cerebellum (sporadic PA) raises the possibility that gliomagenesis requires more than biallelic inactivation of the NF1 tumor suppressor gene or expression of the KIAA1549:BRAF transcript. Several etiologic explanations include differential susceptibilities of preneoplastic neuroglial cell types in different brain regions to these glioma-causing genetic changes, contributions from non-neoplastic cells and signals in the tumor microenvironment, and genomic modifiers that confer glioma risk. As clinically-faithful rodent models of sporadic PA are currently under development, Nf1 genetically-engineered mouse (GEM) models have served as tractable systems to study the role of the cell of origin, deregulated intracellular signaling, non-neoplastic cells in the tumor microenvironment and genomic modifiers in gliomagenesis. In this report, we highlight advances in Nf1-GEM modeling and review new experimental evidence that supports the emerging concept that Nf1- and KIAA1549:BRAF-induced gliomas arise from specific cell types in particular brain locations.

NG2-cells are not the cell of origin for murine neurofibromatosis-1 (Nf1) optic glioma.

Low-grade glial neoplasms (astrocytomas) represent one of the most common brain tumors in the pediatric population. These tumors frequently form in the optic pathway (optic pathway gliomas, OPGs), especially in children with the neurofibromatosis type 1 (NF1)-inherited tumor predisposition syndrome. To model these tumors in mice, we have previously developed several Nf1 genetically-engineered mouse strains that form optic gliomas. However, there are three distinct macroglial cell populations in the optic nerve (astrocytes, NG2+ (nerve/glial antigen 2) cells and oligodendrocytes). The presence of NG2+ cells in the optic nerve raises the intriguing possibility that these cells could be the tumor-initiating cells, as has been suggested for adult glioma. In this report, we used a combination of complementary in vitro and novel genetically-engineered mouse strains in vivo to determine whether NG2+ cells could give rise to Nf1 optic glioma. First, we show that Nf1 inactivation results in a cell-autonomous increase in glial fibrillary acidic protein+ (GFAP+), but not in NG2+, cell proliferation in vitro. Second, similar to the GFAP-Cre transgenic strain that drives Nf1 optic gliomagenesis, NG2-expressing cells also give rise to all three macroglial lineages in vivo. Third, in contrast to the GFAP-Cre strain, Nf1 gene inactivation in NG2+ cells is not sufficient for optic gliomagenesis in vivo. Collectively, these data demonstrate that NG2+ cells are not the cell of origin for mouse optic glioma, and support a model in which gliomagenesis requires Nf1 loss in specific neuroglial progenitors during embryogenesis.

miRNA-145 is downregulated in atypical and anaplastic meningiomas and negatively regulates motility and proliferation of meningioma cells.

Meningiomas are frequent, mostly benign intracranial or spinal tumors. A small subset of meningiomas is characterized by histological features of atypia or anaplasia that are associated with more aggressive biological behavior resulting in increased morbidity and mortality. Infiltration into the adjacent brain tissue is a major factor linked to higher recurrence rates. The molecular mechanisms of progression, including brain invasion are still poorly understood. We have studied the role of micro-RNA 145 (miR-145) in meningiomas and detected significantly reduced miR-145 expression in atypical and anaplastic tumors as compared with benign meningiomas. Overexpression of miR-145 in IOMM-Lee meningioma cells resulted in reduced proliferation, increased sensitivity to apoptosis, reduced anchorage-independent growth and reduction of orthotopic tumor growth in nude mice as compared with control cells. Moreover, meningioma cells with high miR-145 levels had impaired migratory and invasive potential in vitro and in vivo. PCR-array studies of miR145-overexpressing cells suggested that collagen type V alpha (COL5A1) expression is downregulated by miR-145 overexpression. Accordingly, COL5A1 expression was significantly upregulated in atypical and anaplastic meningiomas. Collectively, our data indicate an important anti-migratory and anti-proliferative function of miR-145 in meningiomas.

Parent-of-origin in individuals with familial neurofibromatosis type 1 and optic pathway gliomas.

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant cancer syndromes worldwide. Individuals with NF1 have a wide variety of clinical features including a strongly increased risk for pediatric brain tumors. The etiology of pediatric brain tumor development in NF1 is largely unknown. Recent studies have highlighted the contribution of parent-of-origin effects to tumorigenesis in sporadic cancers and cancer predisposition syndromes; however, there is limited data on this effect for cancers arising in NF1. To increase our understanding of brain tumor development in NF1, we conducted a multi-center retrospective chart review of 240 individuals with familial NF1 who were diagnosed with a pediatric brain tumor (optic pathway glioma; OPG) to determine whether a parent-of-origin effect exists overall or by the patient's sex. Overall, 50 % of individuals with familial NF1 and an OPG inherited the NF1 gene from their mother. Similarly, by sex, both males and females were as likely to inherit the NF1 gene from their mother as from their father, with 52 % and 48 % of females and males with OPGs inheriting the NF1 gene from their mother. In conclusion, in contrast to findings from other studies of sporadic cancers and cancer predisposition syndromes, our results indicate no parent-of-origin effect overall or by patient sex for OPGs in NF1.

Review: low-grade gliomas as neurodevelopmental disorders: insights from mouse models of neurofibromatosis-1.

Over the past few years, the traditional view of brain tumorigenesis has been revolutionized by advances in genomic medicine, molecular biology, stem cell biology and genetically engineered small-animal modelling. We now appreciate that paediatric brain tumours arise following specific genetic mutations in specialized groups of progenitor cells in concert with permissive changes in the local tumour microenvironment. This interplay between preneoplastic/neoplastic cells and non-neoplastic stromal cells is nicely illustrated by the neurofibromatosis type 1-inherited cancer syndrome, in which affected children develop low-grade astrocytic gliomas. In this review, we will use neurofibromatosis type 1 as a model system to highlight the critical role of growth control pathways, non-neoplastic cellular elements and brain region-specific properties in the development of childhood gliomas. The insights derived from examining each of these contributing factors will be instructive in the design of new therapies for gliomas in the paediatric population.

Identification of a progenitor cell of origin capable of generating diverse meningioma histological subtypes.

Meningiomas are among the most common primary central nervous system tumours in adults. Studies focused on the molecular basis for meningioma development are hampered by a lack of information with regard to the cell of origin for these brain tumours. Herein, we identify a prostaglandin D synthase-positive meningeal precursor as the cell of origin for murine meningioma, and show that neurofibromatosis type 2 (Nf2) inactivation in prostaglandin D2 synthase (PGDS) (+) primordial meningeal cells, before the formation of the three meningeal layers, accounts for the heterogeneity of meningioma histological subtypes. Using a unique PGDSCre strain, we define a critical embryonic and early postnatal developmental window in which biallelic Nf2 inactivation in PGDS (+) progenitor cells results in meningioma formation. Moreover, we identify differentially expressed markers that characterize the two major histological meningioma subtypes both in human and mouse tumours. Collectively, these findings establish the cell of origin for these common brain tumours as well as a susceptible developmental period in which signature genetic mutations culminate in meningioma formation.

Regulation of mixed lineage kinase 3 is required for Neurofibromatosis-2-mediated growth suppression in human cancer.

The Neurofibromatosis-2 (NF2) tumor suppressor merlin negatively regulates cell proliferation in numerous cell types. We have previously shown that the NF2 protein (merlin/schwannomin) associates with mixed lineage kinase 3 (MLK3), a mitogen-activated protein kinase (MAPK) kinase kinase that is required for the proliferation of normal and neoplastic cells. In this study, we show that merlin inhibits MLK3 activity, as well as the activation of its downstream effectors, B-Raf, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). The ability of merlin to regulate MLK3 activity requires a direct association between MLK3 and residues in the C-terminal region of merlin. Merlin integrates Rho GTPase family signaling with MAPK activity by inhibiting the binding between MLK3 and its upstream activator, Cdc42. Furthermore, we demonstrate that MLK3 is required for merlin-mediated suppression of cell proliferation and invasion. Collectively, these results establish merlin as a potent inhibitor of MLK3, ERK and JNK activation in cancer, and provide a mechanistic link between deregulated MAPK and Rho GTPase signaling in NF2 growth control.

The ecology of brain tumors: lessons learned from neurofibromatosis-1.

Traditionally, cancer studies have primarily focused on mutations that activate growth or survival pathways in susceptible pre-neoplastic/neoplastic cells. However, recent research has revealed a critical role for non-neoplastic cells within the tumor microenvironment in the process of cancer formation and progression. In addition, the existence of regional and developmental variations in susceptible cell types and supportive microenvironments support a model of tumorigenesis in which the dynamic symbiotic relationship between neoplastic and non-neoplastic cell types dictate where and when cancers form and grow. In this review, we highlight advances in neurofibromatosis type 1 (NF1) genetically engineered mouse brain tumor (glioma) modeling to reveal how cellular and molecular heterogeneity in both the pre-neoplastic/neoplastic and non-neoplastic cellular compartments contribute to gliomagenesis and glioma growth.

Ultrastructural characterization of the optic pathway in a mouse model of neurofibromatosis-1 optic glioma.

The purpose of this study was to investigate the progression of changes in retinal ganglion cells and optic nerve glia in neurofibromatosis-1 (NF1) genetically-engineered mice with optic glioma. Optic glioma tumors were generated in Nf1+/- mice lacking Nf1 expression in GFAP+ cells (astrocytes). Standard immunohistochemistry methods were employed to identify astrocytes (GFAP, S100beta), proliferating progenitor cells (sox2, nestin), microglia (Iba1), endothelial cells (CD31) and retinal ganglion cell (RGC) axons (Neurofilament 68k) in Nf1+/-, Nf1(GFAP)CKO (wild-type mice with Nf1 loss in glial cells), and Nf1+/-(GFAP)CKO (Nf1+/- mice with Nf1 loss in glial cells) mice. Ultrastructural changes in the optic chiasm and nerve were assessed by electron microscopy (EM). RGC were counted in whole retina preparations using high-resolution, mosaic confocal microscopy following their delineation by retrograde FluoroGold labeling. We found that only Nf1+/-(GFAP)CKO mice exhibited gross pre-chiasmatic optic nerve and chiasm enlargements containing aggregated GFAP+/nestin+ and S100beta+/sox2+ cells (neoplastic glia) as well as increased numbers of blood vessels and microglia. Optic gliomas in Nf1+/-(GFAP)CKO mice contained axon fiber irregularities and multilamellar bodies of degenerated myelin. EM and EM tomographic analyses showed increased glial disorganization, disoriented axonal projections, profiles of degenerating myelin and structural alterations at nodes of Ranvier. Lastly, we found reduced RGC numbers in Nf1+/-(GFAP)CKO mice, supporting a model in which the combination of optic nerve Nf1 heterozygosity and glial cell Nf1 loss results in disrupted axonal-glial relationships, subsequently culminating in the degeneration of optic nerve axons and loss of their parent RGC neurons.

Alterations of BRAF and HIPK2 loci predominate in sporadic pilocytic astrocytoma.

OBJECTIVE: Independent studies have previously demonstrated that both the HIPK2 and BRAF genes are amplified and rearranged, respectively, in pilocytic astrocytomas (PAs). The purpose of this study was to further investigate the frequency of BRAF and HIPK2 alterations in PAs, the concordance of these events, and their relationship to clinical phenotype. METHODS: We performed extensive characterization by array-based copy number assessment (aCGH), HIPK2 copy number analysis, and BRAF rearrangement and mutation analysis in a set of 79 PAs, including 9 tumors from patients with neurofibromatosis type 1 (NF1). RESULTS: We identified 1 of 3 previously identified BRAF rearrangements in 42/70 sporadic PAs. An additional 2 tumors with no rearrangement also exhibited BRAF mutation, including a novel 3-base insertion. As predicted from the genomic organization at this locus, 22/36 tumors with BRAF rearrangement also exhibited corresponding HIPK2 amplification. However, 14/36 tumors with BRAF rearrangement had no detectable HIPK2 gene amplification and 6/20 tumors demonstrated HIPK2 amplification without apparent BRAF rearrangement or mutation. Only 12/70 PAs lacked detectable BRAF or HIPK2 alterations. Importantly, none of the 9 PA tumors from NF1 patients exhibited BRAF rearrangement or mutation. CONCLUSIONS: BRAF rearrangement represents the most common genetic alteration in sporadic, but not neurofibromatosis type 1-associated, pilocytic astrocytomas (PAs). These findings implicate BRAF in the pathogenesis of these common low-grade astrocytomas in children, and suggest that PAs arise either from NF1 inactivation or BRAF gain of function.

Expression of the tumor suppressor genes NF2, 4.1B, and TSLC1 in canine meningiomas.

Meningiomas are common primary brain tumors in dogs; however, little is known about the molecular genetic mechanisms involved in their tumorigenesis. Several tumor suppressor genes have been implicated in meningioma pathogenesis in humans, including the neurofibromatosis 2 (NF2), protein 4.1B (4.1 B), and tumor suppressor in lung cancer-1 (TSLC1) genes. We investigated the expression of these tumor suppressor genes in a series of spontaneous canine meningiomas using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) (NF2; n = 25) and western blotting (NF2/merlin, 4.1B, TSLC1; n = 30). Decreased expression of 4.1B and TSLC1 expression on western blotting was seen in 6/30 (20%) and in 15/30 (50%) tumors, respectively, with 18/30 (60%) of meningiomas having decreased or absent expression of one or both proteins. NF2 gene expression assessed by western blotting and RT-PCR varied considerably between individual tumors. Complete loss of NF2 protein on western blotting was not seen, unlike 4.1B and TSLC1. Incidence of TSLC1 abnormalities was similar to that seen in human meningiomas, while perturbation of NF2 and 4.1B appeared to be less common than reported for human tumors. No association was observed between tumor grade, subtype, or location and tumor suppressor gene expression based on western blot or RT-PCR. These results suggest that loss of these tumor suppressor genes is a frequent occurrence in canine meningiomas and may be an early event in tumorigenesis in some cases. In addition, it is likely that other, as yet unidentified, genes play an important role in canine meningioma formation and growth.

The EWSR1/NR4A3 fusion protein of extraskeletal myxoid chondrosarcoma activates the PPARG nuclear receptor gene.

The NR4A3 nuclear receptor is implicated in the development of extraskeletal myxoid chondrosarcoma (EMC), primitive sarcoma unrelated to conventional chondrosarcomas, through a specific fusion with EWSR1 resulting in an aberrant fusion protein that is thought to disrupt the transcriptional regulation of specific target genes. We performed an expression microarray analysis of EMC tumours expressing the EWSR1/NR4A3 fusion protein, comparing their expression profiles to those of other sarcoma types. We thereby identified a set of genes significantly overexpressed in EMC relative to other sarcomas, including PPARG and NDRG2. Western blot or immunohistochemical analyses confirm that PPARG and NDRG2 are expressed in tumours positive for EWSR1/NR4A3. Bioinformatic analysis identified a DNA response element for EWSR1/NR4A3 in the PPARG promoter, and band-shift experiments and transient transfections indicate that EWSR1/NR4A3 can activate transcription through this element. Western blots further show that an isoform of the native NR4A3 receptor lacking the C-terminal domain is very highly expressed in tumours positive for EWSR1/NR4A3, and co-transfections of this isoform along with EWSR1/NR4A3 indicate that it may negatively regulate the activity of the fusion protein on the PPARG promoter. These results suggest that the overall expression of PPARG in EMC may be regulated in part by the balance between EWSR1/NR4A3 and NR4A3, and that PPARG may play a crucial role in the development of these tumours. The specific up-regulation of PPARG by EWSR1/NR4A3 may also have potential therapeutic implications.

High-resolution, dual-platform aCGH analysis reveals frequent HIPK2 amplification and increased expression in pilocytic astrocytomas.

Pilocytic astrocytomas (PAs, WHO grade I) are the most common brain tumors in the pediatric and adolescent population, accounting for approximately one-fifth of central nervous system tumors. Because few consistent molecular alterations have been identified in PAs compared to higher grade gliomas, we performed array comparative genomic hybridization using two independent commercial array platforms. Although whole chromosomal gains and losses were not observed, a 1-Mb amplified region of 7q34 was detected in multiple patient samples using both array platforms. Copy-number gain was confirmed in an independent tumor sample set by quantitative PCR, and this amplification was correlated to both increased mRNA and protein expression of HIPK2, a homeobox-interacting protein kinase associated with malignancy, contained within this locus. Furthermore, overexpression of wild-type HIPK2, but not a kinase-inactive mutant, in a glioma cell line conferred a growth advantage in vitro. Collectively, these results illustrate the power and necessity of implementing high-resolution, multiple-platform genomic analyses to discover small and subtle, but functionally significant, genomic alterations associated with low-grade tumor formation and growth.

Cervical cord compression from plexiform neurofibromas in neurofibromatosis 1.

Cervical cord compression from cervical root neurofibromas represents an important clinical problem in patients with neurofibromatosis type 1 (NF1), but is rarely reported. The aim of this study was to describe the clinical presentation and follow-up of children and adults with NF1 and cervical cord compression. A retrospective review of clinical records and neuroimaging studies from two large tertiary care centres between 1996 and 2006 was performed. 13 patients with NF1 and cervical cord compression were identified. Age at presentation ranged from 9 to 61 years. The most common presentation was progressive quadriparesis. 11 of 13 patients underwent cervical decompression and subtotal resection of the associated neurofibroma. The majority of patients had recovery of neurological function and no further clinical progression. Progressive neurological deficit (typically quadriparesis), rather than neuroimaging appearances, should dictate the need for surgery.

Mammalian target of rapamycin: master regulator of cell growth in the nervous system.

The mammalian target of rapamycin (mTOR) is a highly conserved serine/threonine protein kinase that regulates a number of diverse biologic processes important for cell growth and proliferation, including ribosomal biogenesis and protein translation. In this regard, hyperactivation of the mTOR signaling pathway has been demonstrated in numerous human cancers, including a number of inherited cancer syndromes in which individuals have an increased risk of developing benign and malignant tumors. Three of these inherited cancer syndromes (Lhermitte-Duclos disease, neurofibromatosis type 1, and tuberous sclerosis complex) are characterized by significant central nervous system dysfunction and brain tumor formation. Each of these disorders is caused by a genetic mutation that disrupts the expression of proteins which negatively regulate mTOR signaling, indicating that the mTOR signaling pathway is critical for appropriate brain development and function. In this review, we discuss our current understanding of the mTOR signaling pathway and its role in promoting ribosome biogenesis and cell growth. We suggest that studies of this pathway may prove useful in identifying molecular targets for biologically-based therapies of brain tumors associated with these inherited cancer syndromes as well as sporadic central nervous system tumors.

Tumor suppressor in lung cancer-1 (TSLC1) functions as a glioma tumor suppressor.

Tumor suppressor in lung cancer-1 (TSLC1) loss is common in many human cancers, including meningioma. In this study, we demonstrate that TSLC1 protein and RNA expression is lost in 60% to 65% of high-grade gliomas, and that TSLC1 reintroduction into glioma cells results in growth suppression. Moreover, Tslc1 loss in mice results in increased astrocyte proliferation in vivo and in vitro. These data indicate that TSLC1 functions as a glioma tumor suppressor.