ABSTRACT
Low-grade gliomas are one of the most common brain tumors in children, where they frequently form within the optic pathway (optic pathway gliomas; OPGs). Since many OPGs occur in the context of the Neurofibromatosis Type 1 (NF1) cancer predisposition syndrome, we have previously employed Nf1 genetically-engineered mouse (GEM) strains to study the pathogenesis of these low-grade glial neoplasms. In the light of the finding that human and mouse low-grade gliomas are composed of Olig2+ cells and that Olig2+ oligodendrocyte precursor cells (OPCs) give rise to murine high-grade gliomas, we sought to determine whether Olig2+ OPCs could be tumor-initiating cells for Nf1 optic glioma. Similar to the GFAP-Cre transgenic strain previously employed to generate Nf1 optic gliomas, Olig2+ cells also give rise to astrocytes in the murine optic nerve in vivo. However, in contrast to the GFAP-Cre strain where somatic Nf1 inactivation in embryonic neural progenitor/stem cells (Nf1flox/mut; GFAP-Cre mice) results in optic gliomas by 3 months of age in vivo, mice with Nf1 gene inactivation in Olig2+ OPCs (Nf1flox/mut; Olig2-Cre mice) do not form optic gliomas until 6 months of age. These distinct patterns of glioma latency do not reflect differences in the timing or brain location of somatic Nf1 loss. Instead, they most likely reflect the cell of origin, as somatic Nf1 loss in CD133+ neural progenitor/stem cells during late embryogenesis results in optic gliomas at 3 months of age. Collectively, these data demonstrate that the cell of origin dictates the time to tumorigenesis in murine optic glioma.
Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/pathology , Glioma/genetics , Glioma/pathology , Neurofibromin 1/genetics , Animals , Biomarkers, Tumor , Brain Neoplasms/metabolism , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Gene Expression , Glioma/metabolism , Humans , Immunohistochemistry , Mice , Mice, Knockout , Mice, Transgenic , Mutation , Neoplasm Grading , Neurofibromin 1/metabolism , Oligodendrocyte Transcription Factor 2/genetics , Oligodendrocyte Transcription Factor 2/metabolism , Optic Nerve/metabolism , Optic Nerve/pathologyABSTRACT
Neurofibromatosis type 1 (NF1) is a common neurogenetic condition characterized by significant clinical heterogeneity. A major barrier to developing precision medicine approaches for NF1 is an incomplete understanding of the factors that underlie its inherent variability. To determine the impact of the germline NF1 gene mutation on the optic gliomas frequently encountered in children with NF1, we developed genetically engineered mice harboring two representative NF1-patient-derived Nf1 gene mutations (c.2542G>C;p.G848R and c.2041C>T;p.R681X). We found that each germline Nf1 gene mutation resulted in different levels of neurofibromin expression. Importantly, only R681X(CKO) but not G848R(CKO), mice develop optic gliomas with increased optic nerve volumes, glial fibrillary acid protein immunoreactivity, proliferation and retinal ganglion cell death, similar to Nf1 conditional knockout mice harboring a neomycin insertion (neo) as the germline Nf1 gene mutation. These differences in optic glioma phenotypes reflect both cell-autonomous and stromal effects of the germline Nf1 gene mutation. In this regard, primary astrocytes harboring the R681X germline Nf1 gene mutation exhibit increased basal astrocyte proliferation (BrdU incorporation) indistinguishable from neo(CKO) astrocytes, whereas astrocytes with the G848R mutation have lower levels of proliferation. Evidence for paracrine effects from the tumor microenvironment were revealed when R681X(CKO) mice were compared with conventional neo(CKO) mice. Relative to neo(CKO) mice, the optic gliomas from R681X(CKO) mice had more microglia infiltration and JNK(Thr183/Tyr185) activation, microglia-produced Ccl5, and glial AKT(Thr308) activation. Collectively, these studies establish that the germline Nf1 gene mutation is a major determinant of optic glioma development and growth through by both tumor cell-intrinsic and stromal effects.
Subject(s)
Astrocytes/pathology , Germ-Line Mutation/genetics , Neurofibromatosis 1/complications , Neurofibromin 1/genetics , Optic Nerve Glioma/pathology , Optic Nerve/pathology , Animals , Astrocytes/metabolism , Cells, Cultured , Disease Models, Animal , Female , Glial Fibrillary Acidic Protein/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microglia/metabolism , Microglia/pathology , Optic Nerve/metabolism , Optic Nerve Glioma/etiologyABSTRACT
Neurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder caused by impaired function of the neurofibromin RAS regulator. Using a combination of Nf1 genetically engineered mice and pharmacological/genetic inhibition approaches, we report that neurofibromin differentially controls neural stem cell (NSC) proliferation and multilineage differentiation through the selective use of the PI3K/AKT and RAF/MEK pathways. While PI3K/AKT governs neurofibromin-regulated NSC proliferation, multilineage differentiation is MEK-dependent. Moreover, whereas MEK-regulated multilineage differentiation requires Smad3-induced Jagged-1 expression and Notch activation, MEK/Smad3-regulated Hes1 induction is only responsible for astrocyte and neuronal differentiation. Collectively, these findings establish distinct roles for the RAS effector pathways in regulating brain NSC function.
Subject(s)
Cell Differentiation , Neural Stem Cells/cytology , Neurofibromatosis 1/metabolism , Signal Transduction , ras Proteins/metabolism , Animals , Astrocytes/cytology , Basic Helix-Loop-Helix Transcription Factors/metabolism , Calcium-Binding Proteins/genetics , Cell Lineage , Cell Proliferation , Gene Expression Regulation, Developmental , Homeodomain Proteins/metabolism , Intercellular Signaling Peptides and Proteins/genetics , Jagged-1 Protein , Membrane Proteins/genetics , Mice , Mitogen-Activated Protein Kinase Kinases/metabolism , Neurofibromatosis 1/genetics , Neurons/cytology , Oncogene Protein v-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Receptors, Notch/metabolism , Serrate-Jagged Proteins , Smad3 Protein/genetics , Smad3 Protein/metabolism , Transcription Factor HES-1 , ras Proteins/geneticsABSTRACT
BACKGROUND: Children with neurofibromatosis type 1 (NF1) develop optic pathway gliomas, which result from impaired NF1 protein regulation of Ras activity. One obstacle to the implementation of biologically targeted therapies is an incomplete understanding of the individual contributions of the downstream Ras effectors (mitogen-activated protein kinase kinase [MEK], Akt) to optic glioma maintenance. This study was designed to address the importance of MEK and Akt signaling to Nf1 optic glioma growth. METHODS: Primary neonatal mouse astrocyte cultures were employed to determine the consequence of phosphatidylinositol-3 kinase (PI3K)/Akt and MEK inhibition on Nf1-deficient astrocyte growth. Nf1 optic glioma-bearing mice were used to assess the effect of Akt and MEK inhibition on tumor volume, proliferation, and retinal ganglion cell dysfunction. RESULTS: Both MEK and Akt were hyperactivated in Nf1-deficient astrocytes in vitro and in Nf1 murine optic gliomas in vivo. Pharmacologic PI3K or Akt inhibition reduced Nf1-deficient astrocyte proliferation to wild-type levels, while PI3K inhibition decreased Nf1 optic glioma volume and proliferation. Akt inhibition of Nf1-deficient astrocyte and optic glioma growth reflected Akt-dependent activation of mammalian target of rapamycin (mTOR). Sustained MEK pharmacologic blockade also attenuated Nf1-deficient astrocytes as well as Nf1 optic glioma volume and proliferation. Importantly, these MEK inhibitory effects resulted from p90RSK-mediated, Akt-independent mTOR activation. Finally, both PI3K and MEK inhibition reduced optic glioma-associated retinal ganglion cell loss and nerve fiber layer thinning. CONCLUSION: These findings establish that the convergence of 2 distinct Ras effector pathways on mTOR signaling maintains Nf1 mouse optic glioma growth, supporting the evaluation of pharmacologic inhibitors that target mTOR function in future human NF1-optic pathway glioma clinical trials.
Subject(s)
Brain Neoplasms/metabolism , MAP Kinase Kinase 1/metabolism , Neurofibromatosis 1/metabolism , Optic Nerve Glioma/metabolism , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases/metabolism , Animals , Astrocytes/metabolism , Cell Proliferation , Humans , MAP Kinase Signaling System , Mice , Mice, Inbred C57BL , Neurofibromin 1/genetics , Neurofibromin 1/metabolism , Phosphatidylinositol 3-Kinases/metabolism , TOR Serine-Threonine Kinases/antagonists & inhibitorsABSTRACT
BACKGROUND: Children with the neurofibromatosis type 1 (NF1) tumor predisposition syndrome are prone to the development of optic pathway gliomas resulting from biallelic inactivation of the NF1 gene. Recent studies have revealed the presence of other molecular alterations in a small portion of these NF1-associated brain tumors. The purpose of this study was to leverage Nf1 genetically engineered mouse strains to define the functional significance of these changes to optic glioma biology. METHODS: Nf1+/- mice were intercrossed with Nf1(flox/flox) mice, which were then crossed with Nf1(flox/flox); GFAP-Cre mice, to generate Nf1(flox/mut); GFAP-Cre (FMC) mice. These mice were additionally mated with conditional KIAA1549:BRAF knock-in or Pten(flox/wt) mice to generate Nf1(flox/mut); f-BRAF; GFAP-Cre (FMBC) mice or Nf1(flox/mut); Pten(flox/wt); GFAP-Cre (FMPC) mice, respectively. The resulting optic gliomas were analyzed for changes in tumor volume, proliferation, and retinal ganglion cell loss. RESULTS: While KIAA1549:BRAF conferred no additional biological properties on Nf1 optic glioma, FMPC mice had larger optic gliomas with greater proliferative indices and microglial infiltration. In addition, all 3 Nf1 murine optic glioma strains exhibited reduced retinal ganglion cell survival and numbers; however, FMPC mice had greater retinal nerve fiber layer thinning near the optic head relative to FMC and FMBC mice. CONCLUSIONS: Collectively, these experiments demonstrate genetic cooperativity between Nf1 loss and Pten heterozygosity relevant to optic glioma biology and further underscore the value of employing genetically engineered mouse strains to define the contribution of discovered molecular alterations to brain tumor pathogenesis.
Subject(s)
Neurofibromatosis 1/genetics , Optic Nerve Glioma/genetics , Optic Nerve Neoplasms/genetics , Animals , Cell Death , Disease Models, Animal , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Optic Nerve Glioma/metabolism , Optic Nerve Glioma/pathology , Optic Nerve Neoplasms/metabolism , Optic Nerve Neoplasms/pathology , PTEN Phosphohydrolase/genetics , Retinal Ganglion Cells/pathology , Signal TransductionSubject(s)
Learning Disabilities/etiology , Neurofibromatosis 1/complications , Vision Disorders/etiology , Animals , Female , Humans , MaleABSTRACT
OBJECTIVE: Children with neurofibromatosis-1 (NF1) are at risk for developing numerous nervous system abnormalities, including cognitive problems and brain tumors (optic pathway glioma). Currently, there are few prognostic factors that predict clinical manifestations or outcomes in patients, even in families with an identical NF1 gene mutation. In this study, we leveraged Nf1 genetically engineered mice (GEM) to define the potential role of sex as a clinically relevant modifier of NF1-associated neuronal dysfunction. METHODS: Deidentified clinical data were analyzed to determine the impact of sex on optic glioma-associated visual decline in children with NF1. In addition, Nf1 GEM were employed as experimental platforms to investigate sexually dimorphic differences in learning/memory, visual acuity, retinal ganglion cell (RGC) death, and Nf1 protein (neurofibromin)-regulated signaling pathway function (Ras activity, cyclic adenosine monophosphate [cAMP], and dopamine levels). RESULTS: Female patients with NF1-associated optic glioma were twice as likely to undergo brain magnetic resonance imaging for visual symptoms and 3× more likely to require treatment for visual decline than their male counterparts. As such, only female Nf1 GEM exhibited a decrement in optic glioma-associated visual acuity, shorter RGC axons, and attenuated cAMP levels. In contrast, only male Nf1 GEM showed spatial learning/memory deficits, increased Ras activity, and reduced dopamine levels. INTERPRETATION: Collectively, these observations establish sex as a major prognostic factor underlying neuronal dysfunction in NF1, and suggest that sex should be considered when interpreting future preclinical and clinical study results.
Subject(s)
Learning Disabilities/etiology , Neurofibromatosis 1/complications , Vision Disorders/etiology , Animals , Brain/pathology , Child , Dopamine/metabolism , Dopamine and cAMP-Regulated Phosphoprotein 32/metabolism , Female , Gene Expression Regulation/genetics , Glial Fibrillary Acidic Protein/genetics , Hippocampus/metabolism , Humans , Learning Disabilities/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neurofibromatosis 1/pathology , Neurofibromin 1/genetics , Optic Nerve Glioma/genetics , Sex Factors , Space Perception/physiologyABSTRACT
Low-grade brain tumors (pilocytic astrocytomas) that result from a genomic rearrangement in which the BRAF kinase domain is fused to the amino terminal of the KIAA1549 gene (KIAA1549:BRAF fusion; f-BRAF) commonly arise in the cerebellum of young children. To model this temporal and spatial specificity in mice, we generated conditional KIAA1549:BRAF strains that coexpresses green fluorescent protein (GFP). Although both primary astrocytes and neural stem cells (NSCs) from these mice express f-BRAF and GFP as well as exhibit increased MEK activity, only f-BRAF-expressing NSCs exhibit increased proliferation in vitro. Using Cre driver lines in which KIAA1549:BRAF expression was directed to NSCs (f-BRAF; BLBP-Cre mice), astrocytes (f-BRAF; GFAP-Cre mice), and NG2 progenitor cells (f-BRAF; NG2-Cre mice), increased glial cell numbers were observed only in the cerebellum of f-BRAF; BLBP-Cre mice in vivo. The availability of this unique KIAA1549:BRAF conditional transgenic mouse strain will enable future mechanistic studies aimed at defining the developmentally-regulated temporal and spatial determinants that underlie low-grade astrocytoma formation in children.
Subject(s)
Mice, Transgenic , Neural Stem Cells/physiology , Neuroglia/physiology , Oncogene Proteins, Fusion/physiology , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Animals , Astrocytes/cytology , Astrocytes/physiology , Astrocytoma/genetics , Astrocytoma/pathology , Brain/metabolism , Cell Proliferation , Cerebellum/cytology , Child , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Mice , Mitogen-Activated Protein Kinases/metabolism , Models, Animal , Neuroglia/cytology , Oncogene Proteins, Fusion/genetics , Organ SpecificityABSTRACT
Although traditional models of carcinogenesis have largely focused on neoplastic cells, converging data have revealed the importance of non-neoplastic stromal cells in influencing tumor growth and progression. Leveraging a genetically engineered mouse model of neurofibromatosis type 1 (NF1)-associated optic glioma, we now demonstrate that stromal microglia express the CX3CR1 chemokine receptor, such that reduced CX3CR1 expression decreases optic nerve microglia. Moreover, genetic reduction of Cx3cr1 expression in Nf1 optic glioma mice delays optic glioma formation. Coupled with previous findings demonstrating that microglia maintain optic glioma growth, these new findings provide a strong preclinical rationale for the development of future stroma-directed glioma therapies in children.
Subject(s)
Microglia/physiology , Neurofibromatosis 1/pathology , Optic Nerve Glioma/pathology , Optic Nerve/physiology , Receptors, Chemokine/genetics , Animals , Brain Stem/pathology , Brain Stem/physiology , CX3C Chemokine Receptor 1 , Disease Models, Animal , Mice , Mice, Inbred C57BL , Mice, Knockout , Microglia/pathology , Neurofibromatosis 1/genetics , Neurofibromin 1/genetics , Optic Nerve/pathology , Optic Nerve Glioma/geneticsABSTRACT
The concept that gliomas comprise a heterogeneous group of diseases distinguished by their developmental origin raises the intriguing possibility that neural stem cells (NSCs) from different germinal zones have differential capacities to respond to glioma-causing genetic changes. We demonstrate that lateral ventricle subventricular zone NSCs are molecularly and functionally distinct from those of the third ventricle. Consistent with a unique origin for pediatric low-grade glioma, third ventricle, but not lateral ventricle, NSCs hyperproliferate in response to mutations characteristic of childhood glioma. Finally, we demonstrate that pediatric optic gliomas in Nf1 genetically engineered mice arise from the third ventricle. Collectively, these observations establish the importance of innate brain region NSC heterogeneity in the patterning of gliomagenesis in children and adults.
Subject(s)
Brain Neoplasms/pathology , Glioma/pathology , Neural Stem Cells/pathology , Animals , Child , Genes, Neurofibromatosis 1 , Humans , Mice , Mutation , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Children with the neurofibromatosis-1 (NF1) cancer predisposition syndrome exhibit numerous clinical problems that reflect defective central nervous system (CNS) neuronal function, including learning disabilities, attention deficit disorder, and seizures. These clinical features result from reduced NF1 protein (neurofibromin) expression in NF1+/- (NF1 heterozygosity) brain neurons. Previous studies have shown that mouse CNS neurons are sensitive to the effects of reduced Nf1 expression and exhibit shorter neurite lengths, smaller growth cone areas, and attenuated survival, reflecting attenuated neurofibromin cAMP regulation. In striking contrast, Nf1+/- peripheral nervous system (PNS) neurons are nearly indistinguishable from their wild-type counterparts, and complete neurofibromin loss leads to increased neurite lengths and survival in a RAS/Akt-dependent fashion. To gain insights into the differential responses of CNS and PNS neurons to reduced neurofibromin function, we designed a series of experiments to define the molecular mechanism(s) underlying the unique CNS neuronal sensitivity to Nf1 heterozygosity. First, Nf1 heterozygosity decreases cAMP levels in CNS, but not in PNS, neurons. Second, CNS neurons exhibit Nf1 gene-dependent increases in RAS pathway signaling, but no further decreases in cAMP levels were observed in Nf1-/- CNS neurons relative to their Nf1+/- counterparts. Third, neurofibromin regulates CNS neurite length and growth cone areas in a cAMP/PKA/Rho/ROCK-dependent manner in vitro and in vivo. Collectively, these findings establish cAMP/PKA/Rho/ROCK signaling as the responsible axis underlying abnormal Nf1+/- CNS neuronal morphology with important implications for future preclinical and clinical studies aimed at improving cognitive and behavioral deficits in mice and children with reduced brain neuronal NF1 gene expression.
Subject(s)
Central Nervous System/ultrastructure , Cyclic AMP/metabolism , Heterozygote , Neurofibromin 1/genetics , Neurons/ultrastructure , Signal Transduction/physiology , Animals , Cells, Cultured , Central Nervous System/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Genes, Neurofibromatosis 1 , Growth Cones/ultrastructure , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurites/metabolism , Neurites/ultrastructure , Neurofibromatosis 1/genetics , Neurofibromatosis 1/metabolism , Neurofibromin 1/metabolism , Neurons/cytology , Neurons/metabolism , rho-Associated Kinases/metabolismABSTRACT
Converging evidence from the analysis of human brain tumors and genetically engineered mice has revealed that the mammalian target of rapamycin (mTOR) pathway is a central regulator of glial and glioma cell growth. In this regard, mutational inactivation of neurofibromatosis-1 (NF1), tuberous sclerosis complex (TSC), and PTEN genes is associated with glioma formation, such that pharmacologic inhibition of mTOR signaling results in attenuated tumor growth. This shared dependence on mTOR suggests that PTEN and NF1 (neurofibromin) glial growth regulation requires TSC/Rheb (Ras homolog enriched in brain) control of mTOR function. In this report, we use a combination of genetic silencing in vitro and conditional mouse transgenesis approaches in vivo to demonstrate that neurofibromin regulates astrocyte cell growth and glioma formation in a TSC/Rheb-independent fashion. First, we show that Nf1 or Pten inactivation, but not Tsc1 loss or Rheb overexpression, increases astrocyte cell growth in vitro. Second, Nf1-deficient increased mTOR signaling and astrocyte hyperproliferation is unaffected by Rheb shRNA silencing. Third, conditional Tsc1 inactivation or Rheb overexpression in glial progenitors of Nf1(+/-) mice does not lead to glioma formation. Collectively, these findings establish TSC/Rheb-independent mechanisms for mTOR-dependent glial cell growth control and gliomagenesis relevant to the design of therapies for individuals with glioma.
Subject(s)
Astrocytes/metabolism , Cell Proliferation , Glioma/metabolism , Neurofibromin 1/metabolism , TOR Serine-Threonine Kinases/metabolism , Animals , Astrocytes/cytology , Blotting, Western , Brain/metabolism , Brain/pathology , Female , Glial Fibrillary Acidic Protein/genetics , Glial Fibrillary Acidic Protein/metabolism , Glioma/genetics , Glioma/pathology , HEK293 Cells , Humans , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Monomeric GTP-Binding Proteins/genetics , Monomeric GTP-Binding Proteins/metabolism , Neurofibromin 1/genetics , Neuropeptides/genetics , Neuropeptides/metabolism , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Primary Cell Culture , RNA Interference , Ras Homolog Enriched in Brain Protein , TOR Serine-Threonine Kinases/genetics , Tuberous Sclerosis Complex 1 Protein , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
The identification of mammalian target of rapamycin (mTOR) as a major mediator of neurofibromatosis-1 (NF1) tumor growth has led to the initiation of clinical trials using rapamycin analogs. Previous studies from our laboratory have shown that durable responses to rapamycin treatment in a genetically engineered mouse model of Nf1 optic glioma require 20 mg/kg/day, whereas only transient tumor growth suppression was observed with 5 mg/kg/day rapamycin despite complete silencing of ribosomal S6 activity. To gain clinically relevant insights into the mechanism underlying this dose-dependent effect, we used Nf1-deficient glial cells in vitro and in vivo. First, there was an exponential relationship between blood and brain rapamycin levels. Second, we show that currently used biomarkers of mTOR pathway inhibition (phospho-S6, phospho-4EBP1, phospho-STAT3, and Jagged-1 levels) and tumor proliferation (Ki67) do not accurately reflect mTOR target inhibition or Nf1-deficient glial growth suppression. Third, the incomplete suppression of Nf1-deficient glial cell proliferation in vivo following 5 mg/kg/day rapamycin treatment reflects mTOR-mediated AKT activation, such that combined 5 mg/kg/day rapamycin and PI3-kinase (PI3K) inhibition or dual PI3K/mTOR inhibition recapitulates the growth suppressive effects of 20 mg/kg/day rapamycin. These new findings argue for the identification of more accurate biomarkers for rapamycin treatment response and provide reference preclinical data for comparing human rapamycin levels with target effects in the brain.
Subject(s)
Astrocytes/cytology , Astrocytes/metabolism , Neurofibromatosis 1/genetics , TOR Serine-Threonine Kinases/metabolism , Animals , Antibiotics, Antineoplastic/pharmacology , Astrocytes/drug effects , Brain/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Gene Silencing , Ki-67 Antigen/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Sirolimus/analogs & derivatives , Sirolimus/chemistry , Sirolimus/metabolism , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/geneticsABSTRACT
Whereas carcinogenesis requires the acquisition of driver mutations in progenitor cells, tumor growth and progression are heavily influenced by the local microenvironment. Previous studies from our laboratory have used Neurofibromatosis-1 (NF1) genetically engineered mice to characterize the role of stromal cells and signals to optic glioma formation and growth. Previously, we have shown that Nf1+/- microglia in the tumor microenvironment are critical cellular determinants of optic glioma proliferation. To define the role of microglia in tumor formation and maintenance further, we used CD11b-TK mice, in which resident brain microglia (CD11b+, CD68+, Iba1+, CD45low cells) can be ablated at specific times after ganciclovir administration. Ganciclovir-mediated microglia reduction reduced Nf1 optic glioma proliferation during both tumor maintenance and tumor development. We identified the developmental window during which microglia are increased in the Nf1+/- optic nerve and demonstrated that this accumulation reflected delayed microglia dispersion. The increase in microglia in the Nf1+/- optic nerve was associated with reduced expression of the chemokine receptor, CX3CR1, such that reduced Cx3cr1 expression in Cx3cr1-GFP heterozygous knockout mice led to a similar increase in optic nerve microglia. These results establish a critical role for microglia in the development and maintenance of Nf1 optic glioma.
Subject(s)
Genetic Carrier Screening , Microglia/pathology , Neurofibromatosis 1/genetics , Optic Nerve Glioma/genetics , Optic Nerve Glioma/pathology , Up-Regulation/genetics , Animals , CD11 Antigens/genetics , CX3C Chemokine Receptor 1 , Cell Proliferation , Down-Regulation/genetics , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microglia/metabolism , Receptors, Chemokine/deficiency , Receptors, Chemokine/genetics , Time FactorsABSTRACT
Tumorigenesis requires interactions between tumor progenitors and their microenvironment. We found that low cAMP levels were sufficient for tumorigenesis in a mouse model of Neurofibromatosis-1 (NF1)-associated optic pathway glioma (OPG). We hypothesized that the distinct pattern of glioma in NF1 reflected spatiotemporal differences in CXCL12 effects on cAMP levels. Thus, we sought to alter the pattern of gliomagenesis through manipulation of CXCL12-CXCR4 pathway activation in Nf1 OPG mice. Forced CXCL12 expression induced glioma at a low frequency. Further, treatment of Nf1 OPG mice with AMD3100, a CXCR4 antagonist, did not attenuate glioma growth. Thus, it appears, CXCL12 alone cannot promote gliomagenesis in NF1 mice.
Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Chemokine CXCL12/physiology , Neurofibromin 1/physiology , Optic Nerve Glioma/genetics , Optic Nerve Glioma/metabolism , Animals , Brain Neoplasms/immunology , Cell Line , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/immunology , Cell Transformation, Neoplastic/metabolism , Chemokine CXCL12/biosynthesis , Chemokine CXCL12/genetics , Disease Models, Animal , Gene Expression Regulation, Neoplastic/immunology , Humans , Mice , Mice, Neurologic Mutants , Neurofibromin 1/genetics , Optic Nerve Glioma/immunology , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/immunologyABSTRACT
Current models of oncogenesis incorporate the contributions of chronic inflammation and aging to the patterns of tumor formation. These oncogenic pathways, involving leukocytes and fibroblasts, are not readily applicable to brain tumors (glioma), and other mechanisms must account for microenvironmental influences on central nervous system tumorigenesis. Previous studies from our laboratories have used neurofibromatosis-1 (NF1) genetically engineered mouse (GEM) models to understand the spatial restriction of glioma formation to the optic pathway of young children. Based on our initial findings, we hypothesize that brain region-specific differences in cAMP levels account for the pattern of NF1 gliomagenesis. To provide evidence that low levels of cAMP promote glioma formation in NF1, we generated foci of decreased cAMP in brain regions where gliomas rarely form in children with NF1. Focal cAMP reduction was achieved by forced expression of phosphodiesterase 4A1 (PDE4A1) in the cortex of Nf1 GEM strains. Ectopic PDE4A1 expression produced hypercellular lesions with features of human NF1-associated glioma. Conversely, pharmacologic elevation of cAMP with the PDE4 inhibitor rolipram dramatically inhibited optic glioma growth and tumor size in Nf1 GEM in vivo. Together, these results indicate that low levels of cAMP in a susceptible Nf1 mouse strain are sufficient to promote gliomagenesis, and justify the implementation of cAMP-based stroma-targeted therapies for glioma.
Subject(s)
Cell Transformation, Neoplastic/metabolism , Cyclic AMP/metabolism , Neurofibromatosis 1/metabolism , Optic Nerve Glioma/metabolism , Animals , Brain/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 4/biosynthesis , Cyclic Nucleotide Phosphodiesterases, Type 4/genetics , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Disease Models, Animal , Humans , Mice , Neurofibromatosis 1/genetics , Optic Nerve Glioma/geneticsABSTRACT
Individuals with the neurofibromatosis type 1 (NF1) inherited cancer syndrome exhibit neuronal dysfunction that predominantly affects the CNS. In this report, we demonstrate a unique vulnerability of CNS neurons, but not peripheral nervous system (PNS) neurons, to reduced Nf1 gene expression. Unlike dorsal root ganglion neurons, Nf1 heterozygous (Nf1+/-) hippocampal and retinal ganglion cell (RGC) neurons have decreased growth cone areas and neurite lengths, and increased apoptosis compared to their wild-type counterparts. These abnormal Nf1+/- CNS neuronal phenotypes do not reflect Ras pathway hyperactivation, but rather result from impaired neurofibromin-mediated cAMP generation. In this regard, elevating cAMP levels with forskolin or rolipram treatment, but not MEK (MAP kinase kinase) or PI3-K (phosphatidylinositol 3-kinase) inhibition, reverses these abnormalities to wild-type levels in vitro. In addition, Nf1+/- CNS, but not PNS, neurons exhibit increased apoptosis in response to excitotoxic or oxidative stress in vitro. Since children with NF1-associated optic gliomas often develop visual loss and Nf1 genetically engineered mice with optic glioma exhibit RGC neuronal apoptosis in vivo, we further demonstrate that RGC apoptosis resulting from optic glioma in Nf1 genetically engineered mice is attenuated by rolipram treatment in vivo. Similar to optic glioma-induced RGC apoptosis, the increased RGC neuronal death in Nf1+/- mice after optic nerve crush injury is also attenuated by rolipram treatment in vivo. Together, these findings establish a distinctive role for neurofibromin in CNS neurons with respect to vulnerability to injury, define a CNS-specific neurofibromin intracellular signaling pathway responsible for neuronal survival, and lay the foundation for future neuroprotective glioma treatment approaches.
Subject(s)
Central Nervous System/metabolism , Cyclic AMP/deficiency , Genetic Carrier Screening , Neurofibromatosis 1/genetics , Neurofibromatosis 1/metabolism , Neurons/metabolism , Animals , Apoptosis/genetics , Cell Survival/genetics , Cells, Cultured , Central Nervous System/pathology , Cyclic AMP/biosynthesis , Cyclic AMP/genetics , Hippocampus/metabolism , Hippocampus/pathology , Mice , Mice, Knockout , Nerve Crush , Neurons/pathology , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/pathologyABSTRACT
Neurofibromatosis type 1 (NF1) is a common cancer predisposition syndrome in which affected individuals develop benign and malignant nerve tumors. The NF1 gene product neurofibromin negatively regulates Ras and mammalian target of rapamycin (mTOR) signaling, prompting clinical trials to evaluate the ability of Ras and mTOR pathway inhibitors to arrest NF1-associated tumor growth. To discover other downstream targets of neurofibromin, we performed an unbiased cell-based high-throughput chemical library screen using NF1-deficient malignant peripheral nerve sheath tumor (MPNST) cells. We identified the natural product, cucurbitacin-I (JSI-124), which inhibited NF1-deficient cell growth by inducing apoptosis. We further showed that signal transducer and activator of transcription-3 (STAT3), the target of cucurbitacin-I inhibition, was hyperactivated in NF1-deficient primary astrocytes and neural stem cells, mouse glioma cells, and human MPNST cells through Ser(727) phosphorylation, leading to increased cyclin D1 expression. STAT3 was regulated in NF1-deficient cells of murine and human origin in a TORC1- and Rac1-dependent manner. Finally, cucurbitacin-I inhibited the growth of NF1-deficient MPNST cells in vivo. In summary, we used a chemical genetics approach to reveal STAT3 as a novel neurofibromin/mTOR pathway signaling molecule, define its action and regulation, and establish STAT3 as a tractable target for future NF1-associated cancer therapy studies.
Subject(s)
Cell Proliferation , Neurofibromin 1/physiology , STAT3 Transcription Factor/metabolism , Animals , Animals, Newborn , Cell Proliferation/drug effects , Cells, Cultured , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Genes, Tumor Suppressor/physiology , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Male , Mice , Mice, Nude , Mice, Transgenic , Morpholines/pharmacology , Neurofibromin 1/genetics , Protein Serine-Threonine Kinases/metabolism , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/physiology , Signal Transduction/drug effects , Signal Transduction/genetics , TOR Serine-Threonine Kinases , Triterpenes/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Numerous studies have suggested that astrocytes in the central nervous system (CNS) exhibit molecular and functional heterogeneity. In this regard, astroglia from different CNS locations express distinct immune system, and neurotransmitter proteins, have varying levels of gap junction coupling and respond differently to injury. However, the relevance of these differences to human disease is unclear. As brain tumors in children arise in specific CNS locations, we hypothesized that regional astroglial cell heterogeneity might partly underlie the propensity for gliomas to arise in these areas. In this study, we performed high-density RNA microarray profiling on astrocytes from postnatal day 1 optic nerve, cerebellum, brainstem, and neocortex. We showed that astroglia from each region are molecularly distinct, and we were able to develop gene expression patterns that distinguish astroglia, but not neural stem cells, from these different brain regions. We next used these microarray data to determine whether brain tumor suppressor genes were differentially expressed in these distinct populations of astroglia. Interestingly, neurofibromatosis type 1 (NF1) gene expression was decreased at both the RNA and protein levels in neocortical astroglia relative to astroglia from the other brain regions. To determine the functional significance of this finding, we found increased astroglial cell proliferation in optic nerve, brainstem, and cerebellum, but not neocortex, following Nf1 inactivation in vitro and in vivo. These findings provide molecular evidence for CNS astroglial cell heterogeneity, and suggest that differences in tumor suppressor gene expression might contribute to the regional localization of human brain tumors.
Subject(s)
Astrocytes/cytology , Astrocytes/metabolism , Brain/metabolism , Cell Proliferation , Neurofibromin 1/metabolism , Animals , Animals, Newborn , Brain/cytology , Brain Stem/metabolism , Cells, Cultured , Cerebellum/metabolism , Gene Expression , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neocortex/metabolism , Neurofibromin 1/genetics , Neurons/metabolism , Oligonucleotide Array Sequence Analysis , Optic Nerve/metabolism , Stem Cells/metabolismABSTRACT
Neurofibromatosis-1 (NF1) is a common tumor predisposition syndrome in which affected individuals develop benign and malignant tumors. Previous studies from our laboratory and others have shown that benign tumor formation in Nf1 genetically engineered mice (GEM) requires a permissive tumor microenvironment. In the central nervous system, Nf1 loss in glia is insufficient for glioma formation unless coupled with Nf1 heterozygosity in the brain. Our subsequent studies identified Nf1+/- microglia as a critical cellular determinant of optic glioma growth in Nf1 GEM. Using NF1 as an experimental paradigm to further characterize the role of microglia in glioma growth, we first examined the properties of Nf1+/- microglia in vitro and in vivo. Nf1+/- microglia exhibit increased proliferation and motility and express elevated levels of genes associated with microglia activation. We further show that Nf1+/- microglia harbor high levels of activated c-Jun-NH(2)-kinase (JNK) without any significant changes in Akt, mitogen-activated protein kinase (MAPK), or p38-MAPK activity. In contrast, Nf1-/- astrocytes do not exhibit increased JNK activation. SP600125 inhibition of JNK activity in Nf1+/- microglia results in amelioration of the increased proliferation and motility phenotypes and reduces the levels of expression of activated microglia-associated transcripts. Moreover, SP600125 treatment of Nf1 optic glioma-bearing GEM results in reduced optic glioma proliferation in vivo. Collectively, these findings suggest that Nf1+/- microglia represent a good model system to study the role of specialized microglia in brain tumorigenesis and identify a unique Nf1 deregulated pathway for therapeutic studies aimed at abrogating microenvironmental signals that promote brain tumor growth.