Balazs Hegedus

Neurofibromatosis-1 Regulates Neuronal and Glial Cell Differentiation from Neuroglial Progenitors In Vivo by Both cAMP- and Ras-Dependent Mechanisms

Individuals with neurofibromatosis type 1 (NF1) develop abnormalities of both neuronal and glial cell lineages, suggesting that the NF1 protein neurofibromin is an essential regulator of neuroglial progenitor function. In this regard, Nf1-deficient embryonic telencephalic neurospheres exhibit increased self-renewal and prolonged survival as explants in vivo. Using a newly developed brain lipid binding protein (BLBP)-Cre mouse strain to study the role of neurofibromin in neural progenitor cell function in the intact animal, we now show that neuroglial progenitor Nf1 inactivation results in increased glial lineage proliferation and abnormal neuronal differentiation in vivo. Whereas the glial cell lineage abnormalities are recapitulated by activated Ras or Akt expression in vivo, the neuronal abnormalities were Ras- and Akt independent and reflected impaired cAMP generation in Nf1-deficient cells in vivo and in vitro. Together, these findings demonstrate that neurofibromin is required for normal glial and neuronal development involving separable Ras-dependent and cAMP-dependent mechanisms.

Encephalitis and GABAB receptor antibodies: Novel findings in a new case series of 20 patients

Objective: To report the clinical features of 20 newly diagnosed patients with GABAB receptor (GABABR) antibodies and determine the frequency of associated tumors and concurrent neuronal autoantibodies. Methods: Clinical data were retrospectively obtained and evaluated. Serum and CSF samples were examined for additional antibodies using methods previously reported. Results: Seventeen patients presented with seizures, memory loss, and confusion, compatible with limbic encephalitis (LE), one patient presented with ataxia, one patient presented with status epilepticus, and one patient presented with opsoclonus-myoclonus syndrome (OMS). Nineteen (95%) patients eventually developed LE during the course of the disease. Small-cell lung cancer (SCLC) was identified in 10 (50%) patients, all with LE. Treatment and outcome was available from 19 patients: 15 showed complete (n = 7) or partial (n = 8) neurologic improvement after steroids, IV immunoglobulins, or plasma exchange and oncologic treatment when indicated; 1 patient died of tumor progression shortly after the first cycle of immunotherapy, and 3 were not treated. Five patients with SCLC had additional onconeuronal antibodies (Ri, amphiphysin, or SOX1), and 2 without tumor had GAD65 and NMDAR antibodies, respectively. GABABR antibodies were not detected in serum of 116 patients with SCLC without neurologic symptoms. Conclusion: Our study confirms GABABR as an autoantigen of paraneoplastic and nonparaneoplastic LE and expands the phenotype of GABABR antibodies to ataxia, OMS, and status epilepticus. The long-term prognosis is dictated by the presence of a tumor. Recognition of syndromes associated with GABABR antibodies is important because they usually respond to treatment.

Preclinical cancer therapy in a mouse model of neurofibromatosis-1 optic glioma.

Mouse models of human cancers afford unique opportunities to evaluate novel therapies in preclinical trials. For this purpose, we analyzed three genetically engineered mouse (GEM) models of low-grade glioma resulting from either inactivation of the neurofibromatosis-1 (Nf1) tumor suppressor gene or constitutive activation of KRas in glial cells. Based on tumor proliferation, location, and penetrance, we selected one of these Nf1 GEM models for preclinical drug evaluation. After detection of an optic glioma by manganese-enhanced magnetic resonance imaging, we randomized mice to either treatment or control groups. We first validated the Nf1 optic glioma model using conventional single-agent chemotherapy (temozolomide) currently used for children with low-grade glioma and showed that treatment resulted in decreased proliferation and increased apoptosis of tumor cells in vivo as well as reduced tumor volume. Because neurofibromin negatively regulates mammalian target of rapamycin (mTOR) signaling, we showed that pharmacologic mTOR inhibition in vivo led to decreased tumor cell proliferation in a dose-dependent fashion associated with a decrease in tumor volume. Interestingly, no additive effect of combined rapamycin and temozolomide treatment was observed. Lastly, to determine the effect of these therapies on the normal brain, we showed that treatments that affect tumor cell proliferation or apoptosis did not have a significant effect on the proliferation of progenitor cells within brain germinal zones. Collectively, these findings suggest that this Nf1 optic glioma model may be a potential preclinical benchmark for identifying novel therapies that have a high likelihood of success in human clinical trials.

Nucleophosmin Mediates Mammalian Target of Rapamycin–Dependent Actin Cytoskeleton Dynamics and Proliferation in Neurofibromin-Deficient Astrocytes

Neurofibromatosis type 1 (NF1) is a common autosomal dominant tumor predisposition syndrome in which affected individuals develop astrocytic brain tumors (gliomas). To determine how the NF1 gene product (neurofibromin) regulates astrocyte growth and motility relevant to glioma formation, we have used Nf1-deficient primary murine astrocytes. Nf1(-/-) astrocytes exhibit increased protein translation and cell proliferation, which are mediated by Ras-dependent hyperactivation of the mammalian target of rapamycin (mTOR) protein, a serine/threonine protein kinase that regulates ribosomal biogenesis, protein translation, actin cytoskeleton dynamics, and cell proliferation. In this study, we show that Nf1-deficient astrocytes have fewer actin stress fibers and exhibit increased cell motility compared with wild-type astrocytes, which are rescued by pharmacologic and genetic mTOR inhibition. We further show that mTOR-dependent regulation of actin stress fiber formation, motility, and proliferation requires rapamycin-sensitive activation of the Rac1 GTPase but not elongation factor 4E-binding protein 1/S6 kinase. Nf1(-/-) astrocytes also exhibit increased protein translation and ribosomal biogenesis through increased expression of the nucleophosmin (NPM) nuclear-cytoplasmic shuttling protein. We found that NPM expression in Nf1(-/-) astrocytes was blocked by rapamycin in vitro and in vivo and that expression of a dominant-negative NPM mutant protein in Nf1(-/-) astrocytes rescued actin stress fiber formation and restored cell motility and proliferation to wild-type levels. Together, these data show that neurofibromin regulates actin cytoskeleton dynamics and cell proliferation through a mTOR/Rac1-dependent signaling pathway and identify NPM as a critical mTOR effector mediating these biological properties in Nf1-deficient astrocytes.

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.

The neurobiology of neurooncology.

The histological classification of brain tumors currently is based on the morphological appearance and protein expression patterns that reflect specific cell types within the central nervous system. Recent studies have suggested that the cells of origin for brain tumors may persist in the fully formed tumors, and that these “cancer stem cells” might represent the relevant cellular targets for anticancer therapy. In this regard, insights into the developmental neurobiology of brain tumors has significant impact on our understanding of the molecular and cellular pathogenesis of these devastating cancers, as well as the development of new strategies for treating brain tumors. Ann Neurol 2006;60:3–11

Apelin Expression in Human Non-small Cell Lung Cancer: Role in Angiogenesis and Prognosis

Introduction: The recently discovered bioactive peptide, apelin, has been demonstrated to stimulate angiogenesis in various experimental systems. However, its clinical significance and role in tumor vascularization have not yet been investigated in a human malignancy. Therefore, our aim was to study whether apelin expression is associated with angiogenesis and/or tumor growth/behavior in human non-small cell lung cancer (NSCLC). Methods: A total of 94 patients with stage I–IIIA NSCLC and complete follow-up information were included. Apelin expression in human NSCLC samples and cell lines was measured by quantitative reverse-transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. Effects of exogenous apelin and apelin transfection were studied on NSCLC cell lines in vitro. In vivo growth of tumors expressing apelin or control vectors were also assessed. Morphometric variables of human and mouse tumor capillaries were determined by anti-CD31 labeling. Results: Apelin was expressed in all of the six investigated NSCLC cell lines both at the mRNA and protein levels. Although apelin overexpression or apelin treatments did not increase NSCLC cell proliferation in vitro, increasing apelin levels by gene transfer to NSCLC cells significantly stimulated tumor growth and microvessel densities and perimeters in vivo. Apelin mRNA levels were significantly increased in human NSCLC samples compared with normal lung tissue, and high apelin protein levels were associated with elevated microvessel densities and poor overall survival. Conclusions: This study reveals apelin as a novel angiogenic factor in human NSCLC. Moreover, it also provides the first evidence for a direct association of apelin expression with clinical outcome in a human cancer.

Optic Nerve Dysfunction in a Mouse Model of Neurofibromatosis-1 Optic Glioma

Individuals with neurofibromatosis type 1 (NF1) are prone to developoptic pathway gliomas that can result in significant visual impairment. To explore the cellular basis for the reduced visual functionresulting from optic glioma formation, we used a genetically engineered mouse model of Nf1 optic glioma (Nf1+/−GFAPCKO mice). We performed multimodal functional and structural analyses both before and after the appearance of macroscopic tumors. At 6weeks of age, before obvious glioma formation, Nf1+/−GFAPCKO mice had decreased visual-evoked potential amplitudes and increased optic nerve axon calibers. By 3 months of age, Nf1+/−GFAPCKO mice exhibited pronounced optic nerve axonopathy and apoptosis ofneurons in the retinal ganglion cell layer. Magnetic resonance diffusion tensor imaging showed a progressive increase in radial diffusivity between 6 weeks and 6 months of age in the optic nerve proximal to the tumor indicating ongoing deterioration of axons. These data suggest that optic glioma formation results in early axonaldisorganization and damage, which culminates in retinal ganglion cell death. Collectively, this study shows that Nf1+/−GFAPCKO mice can provide a useful model for defining mechanisms of visual abnormalities in children with NF1 and lay the foundations for future interventional studies aimed at reducing visual loss.

Irradiation and Taxol treatment result in non-monotonous, dose-dependent changes in the motility of glioblastoma cells.

AbstractObjective: Three-dimensional conformal radiotherapy, stereotactic radiosurgery and concurrent chemoradiotherapy are among the most important postoperative therapeutic measures in the treatment of malignant gliomas. We investigated in vitro how these modalities affect cell motility, a key factor in tumor invasiveness and malignancy. Methods: A highly motile glioblastoma cell line was exposed to clinically relevant (2–20 Gy) radiation doses. Some cultures were also subjected to radiosensitizing treatment, in which 5 and 10 nM Taxol was added to the medium for 2 h before the irradiation. The surviving cell fraction was continuously monitored during a 3 day-long time period using an automatized scanning videomicroscope system. Cell motility on a two-dimensional substrate was analyzed by following a large population of cells in each culture. Average velocities, their distribution within the population and persistence of migration were calculated from the cell trajectories. Results: Irradiation increases both the persistence of migration and the heterogeneity of the cell population. Moreover, it results in a non-monotonous alteration of cell motility: While >10 Gy doses impair motion, exposure to 2 Gy increases velocities by 20%. Taxol treatment reduced the motility of irradiated cells, while slightly increased the velocities of non-irradiated cells. We thus show that – at least for certain glioblastoma cells – both irradiation and Taxol treatment can substantially and synergistically influence cell motility. Conclusions: High grade gliomas are characterized by bad prognosis and poor response to therapy. The unexpected motogenic effect of low-dose radiation and paclitaxel treatments highlight the importance of similar investigations to develop more effective clinical treatments.

Vessel co-option is common in human lung metastases and mediates resistance to anti-angiogenic therapy in preclinical lung metastasis models

Anti‐angiogenic therapies have shown limited efficacy in the clinical management of metastatic disease, including lung metastases. Moreover, the mechanisms via which tumours resist anti‐angiogenic therapies are poorly understood. Importantly, rather than utilizing angiogenesis, some metastases may instead incorporate pre‐existing vessels from surrounding tissue (vessel co‐option). As anti‐angiogenic therapies were designed to target only new blood vessel growth, vessel co‐option has been proposed as a mechanism that could drive resistance to anti‐angiogenic therapy. However, vessel co‐option has not been extensively studied in lung metastases, and its potential to mediate resistance to anti‐angiogenic therapy in lung metastases is not established. Here, we examined the mechanism of tumour vascularization in 164 human lung metastasis specimens (composed of breast, colorectal and renal cancer lung metastasis cases). We identified four distinct histopathological growth patterns (HGPs) of lung metastasis (alveolar, interstitial, perivascular cuffing, and pushing), each of which vascularized via a different mechanism. In the alveolar HGP, cancer cells invaded the alveolar air spaces, facilitating the co‐option of alveolar capillaries. In the interstitial HGP, cancer cells invaded the alveolar walls to co‐opt alveolar capillaries. In the perivascular cuffing HGP, cancer cells grew by co‐opting larger vessels of the lung. Only in the pushing HGP did the tumours vascularize by angiogenesis. Importantly, vessel co‐option occurred with high frequency, being present in >80% of the cases examined. Moreover, we provide evidence that vessel co‐option mediates resistance to the anti‐angiogenic drug sunitinib in preclinical lung metastasis models. Assuming that our interpretation of the data is correct, we conclude that vessel co‐option in lung metastases occurs through at least three distinct mechanisms, that vessel co‐option occurs frequently in lung metastases, and that vessel co‐option could mediate resistance to anti‐angiogenic therapy in lung metastases. Novel therapies designed to target both angiogenesis and vessel co‐option are therefore warranted.