Granule Neuron Precursors Research Articles

Effects of altered ephrin-A5 and EphA4/EphA7 expression on tumor growth in a medulloblastoma mouse model.

BackgroundMembers of the Eph/ephrin gene families act as key regulators of cerebellar development during embryogenesis. Aberrant signaling of Eph family of receptor tyrosine kinases and their ephrin ligands has also been implicated in human cancers. Medulloblastoma is an aggressive primitive neuroectodermal tumor that originates from granule neuron precursors in the cerebellum. Previous studies have suggested a role for the ephrin-A5 ligand and its receptors, EphA4 and EphA7, in granule cell-precursor formation and in guiding cell migration. In the present study, we investigated the effects of genetic loss of ephrin-A5, EphA4, and EphA7 on the spatiotemporal development of medulloblastoma tumors in the context of the smoothened transgenic mouse model system.FindingsRadiographic magnetic resonance imaging (MRI) was performed to monitor tumor growth in a genetically engineered mouse model of medulloblastoma. Tumor tissue was harvested to determine changes in the expression of phosphorylated Akt by Western blotting. This helped to establish a correlation between genotype and/or tumor size and survival. Our in vivo data establish that in ND2-SmoA1 transgenic mice, the homozygous deletion of ephrin-A5 resulted in a consistent pattern of tumor growth inhibition compared to their ephrin-A5 wild-type littermate controls, while the loss of EphA4/EphA7 failed to produce consistent effects versus EphA4/EphA7 wild-type mice. A positive correlation was evident between tumor size, p-Akt, and proliferating cell nuclear antigen (PCNA) expression in our transgenic mouse model system, regardless of genotype.ConclusionsTaken together, our findings underscore the importance of targeting specific members of the Eph/ephrin families in conjunction with the Akt pathway in order to inhibit medulloblastoma tumor growth and progression.

Aryl hydrocarbon receptor deletion in cerebellar granule neuron precursors impairs neurogenesis.

The aryl hydrocarbon receptor (AhR) is a ligand-activated member of the basic-helix-loop-helix/PER-ARNT-SIM(PAS) transcription factor superfamily that also mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increasing evidence suggests that AhR influences the development of many tissues, including the central nervous system. Our previous studies suggest that sustained AhR activation by TCDD and/or AhR deletion disrupts cerebellar granule neuron precursor (GNP) development. In the current study, to determine whether endogenous AhR controls GNP development in a cell-autonomous manner, we created a GNP-specific AhR deletion mouse, AhR(fx/fx) /Math1(CRE/+) (AhR CKO). Selective AhR deletion in GNPs produced abnormalities in proliferation and differentiation. Specifically, fewer GNPs were engaged in S-phase, as demonstrated by ∼25% reductions in thymidine (in vitro) and Bromodeoxyuridine (in vivo) incorporation. Furthermore, total granule neuron numbers in the internal granule layer at PND21 and PND60 were diminished in AhR conditional knockout (CKO) mice compared with controls. Conversely, differentiation was enhanced, including ∼40% increase in neurite outgrowth and 50% increase in GABARα6 receptor expression in deletion mutants. Our results suggest that AhR activity plays a role in regulating granule neuron number and differentiation, possibly by coordinating this GNP developmental transition. These studies provide novel insights for understanding the normal roles of AhR signaling during cerebellar granule cell neurogenesis and may have important implications for the effects of environmental factors in cerebellar dysgenesis.

NrCAM modulates sonic hedgehog signalling by controlling smoothened translocation in the cilium

Objective: Cerebellar development involves a spurt of proliferation in external granule layer (EGL) in response to shh, causing granule neuron precursors (GNPs) to proliferate. These cells subsequently differentiate into granule neurons in the inner granule layer (IGL). F3, a CNTN family molecule, can interact with NrCAM to switch GNPs from proliferation to differentiation. We aim to identify the role of NrCAM in the sonic hedgehog response in GNPs. Methods: GNPs were extracted from wildtype and NrCAM mutant P5 cerebella using Percoll gradient centrifugation. Proliferation response to shh was measured using EdU in presence/absence of F3-Fc. GNPs treated with shh/SAG were stained with antibodies against Arl13b and smo to look for differences in cilia size and smo occupancy after different treatment times. Results: NrCAM-/- and wildtype GNPs both proliferated equally in response to shh. F3 was found to block the proliferation response in wildtype but not in NrCAM-/- GNPs. F3 also failed to affect proliferation in SmoA1 GNPs with a constitutively active smo suggesting that the F3-NrCAM mediated block lay upstream of Smo. NrCAM was detected in wildtype cilia and Smo localization was affected in NrCAM-/- GNPs. No differences in cilia length were observed. Conclusion: Our results suggest that NrCAM affects shh-mediated proliferation by controlling smo movement into the cilium.

Genetic depletion of cyclin dependent Kinase-5 results in enhanced tumor rejection via a CD4+ T cell dependent mechanism (TUM7P.1023)

Abstract CDK5 is a non-canonical member of the CDK family that does not require cell cycling for activity but relies on binding to its obligate activators, p35 and p39 which are mostly expressed in post mitotic neurons. CDK5 is required for normal Central Nervous System (CNS) development. Medulloblastoma (MB), derived from primitive cerebellar granule neuron precursors, is the most common pediatric CNS malignancy. Utilizing a short hairpin RNA approach to silence Cdk5 expression in a C57BL/6 (B6) syngenic MB line derived from p53+/- PTCH-/- (MM1), we found that 19 days after subcutaneous inoculation, mice with silenced Cdk5 (MM1-CDK5-KD) revealed a 78% tumor free survival (N=36) compared to 4% (N=24) of mice inoculated with wild type MB (MM1-WT). Furthermore, when injected into Nu/Nu mice, MM1-WT and MM1-CDK5-KD had similar subcutaneous growth kinetics. When CD4+ cells were depleted in B6 mice inoculated with MM1-CDK5-KD tumor free survival decreased to 28% (N=15). We sought to interrogate the role of the immune system in enhanced killing of MM1-CDK5-KD tumors. When exposed to IFN-γ stimulation in vitro, genetic profiling of MM1-CDK5-KD revealed a decrease in PD-L1 expression, a known immune checkpoint surface molecule, compared to MM1-WT. These results indicate a functional role for Cdk5 in MB immune evasion which, when circumvented via Cdk5 inhibition, leads to increased susceptibility to anti-tumor surveillance.

MB-36 * WIP1 AUGMENTS SONIC HEDGEHOG (SHH) SIGNALING AND IS A TARGET IN SHH MEDULLOBLASTOMAS

Sonic Hedgehog (SHH) signaling is active in 30% of childhood medulloblastomas. Dysregulation of p53 signaling is especially prognostic for poor survival in SHH medulloblastoma. But, p53 is mutated in <10% of medulloblastomas. Genomic analyses have demonstrated high-level amplification of PPM1D (WIP1) in p53 wild-type SHH medulloblastomas. WIP1 functions as an oncogene, in part by inactivating p53. We hypothesized that, in the absence of p53 mutation, WIP1 overexpression augments SHH-driven medulloblastoma tumorigenesis and is an important target in the treatment of SHH-active medulloblastomas. WIP1 overexpression in murine granule neuron precursor (GNP) cells enhanced expression of Shh target genes, increased the percentage of cells in S phase, and increased cell viability in response to Shh stimulation. Although WIP1 is known to primarily act through p53 pathways, we found that the interaction between WIP1 and Shh was partially p53-independent. To further understand WIP1 mechanisms in vivo, we developed a mouse in which WIP1 is expressed in the developing brain under control of the Neurod2 promoter (ND2:WIP1). The external granule layer in early post-natal ND2:WIP1 mice exhibited increased proliferation and expression of Shh downstream targets. When ND2:WIP1 transgenic mice were crossed with SmoA1/SmoA1 medulloblastoma-prone mice, medulloblastoma incidence increased and double transgenic SmoA1/SmoA1; ND2:WIP1+ mice demonstrated reduced survival. Conversely, Wip1 knock out significantly suppressed medulloblastoma formation in SmoA1/SmoA1 as well as Tamoxifen-treated Math1-creER; Ptc1 fl/fl mice. Treatment with a lentivirus that knocks down Wip1 or with the WIP1 inhibitor CCT007093 inhibits the effects of Shh stimulation and potentiates the effects of the SHH pathway-inhibiting drugs SANT-1, cyclopamine, or the clinically-relevant LDE225 on the growth of either SmoA1/SmoA1 or Math1-creER + ; Ptc1 fl/fl medulloblastoma cells in vitro. This suggests an important cross-talk between SHH and WIP1 that accelerates tumorigenesis. Our findings also suggest an important role for WIP1 inhibition in the treatment of SHH-active medulloblastomas.

BI-04 * PID1 IS A CHEMOTHERAPY SENSITIZER AND CANDIDATE TUMOR SUPPRESSOR IN MEDULLOBLASTOMA

We recently showed that medulloblastoma PID1 mRNA was highly correlated with survival (Erdreich-Epstein et al, Clinical Cancer Research 2014). PID1 mRNA was lower in Group 3 and Group 4 medulloblastoma and higher in SHH medulloblastoma. In cultured medulloblastoma cells we also showed that PID1 expression induced apoptosis, decreased proliferation and suppressed phosphorylation of AKT and ERK. PID1 also induced depolarization of mitochondrial membrane potential, while siRNA to PID1 decreased it. We now show that PID1 is growth inhibitory to medulloblastoma tumors in vivo: cerebellar granule neuron precursors from pups of our BarTeL Tva-based genetically-engineered mouse that were transduced ex vivo with Pid1/Shh/Mycn and injected into mouse cerebella grew slower than those transduced only with Shh/Mycn. This indicates that in addition to its growth inhibitory effects in culture, PID1 is also inhibitory to medulloblastoma growth in vivo. We hypothesized that the improved survival in patients whose tumors had higher PID1 mRNA reflected a higher sensitivity of these tumors to therapy. Indeed, in culture, transient transfection of PID1 augmented etoposide- and cisplatin-induced apoptosis compared to vector control combined with these drugs. Moreover, siRNA knockdown of PID mitigated the chemotherapy-induced apoptosis, indicating that some of the chemotherapy effect was mediated by PID1. Interestingly, cisplatin, etoposide and vincristine also increased PID1 mRNA in medulloblastoma cells, and this increase was blocked by NFkappaB inhibitors, BAY-117082 and JSH-23. Taken together, our data indicate that PID1 is a candidate tumor suppressor for medulloblastoma, and that it sensitizes medulloblastoma to chemotherapy in an NFkappaB-dependent manner.

SG-03 * A ROLE FOR NADPH OXIDASE 4-GENERATED REACTIVE OXYGEN SPECIES IN SONIC HEDGEHOG-DRIVEN PROLIFERATION OF CEREBELLAR GRANULE NEURON PRECURSORS

Medulloblastoma is the most common solid malignant pediatric brain tumor. These tumors arise in the cerebellum and can be molecularly subdivided into 4 consensus subgroups, one of which is marked by amplification and activation of Sonic hedgehog (Shh) pathway components and downstream targets. This subclass is proposed to arise from the oncogenic transformation of cerebellar granule neuron precursors (CGNPs), whose expansion during post-natal brain development requires activation of the Shh pathway and downstream targets. These tumors often demonstrate similarities with normal cerebellar development at the molecular level, thus allowing us to use primary CGNP cultures as a model system for the Sonic hedgehog (Shh) driven subclass of medulloblastoma. In addition to mitogens driving proliferation in cancer, it has been shown in the past that low levels of intracellular reactive oxygen species (ROS) can contribute to proliferation through, amongst other methods, phosphatase inhibition and subsequent deregulation of key pathways including pathways that collaborate with Shh signaling. To this end we've studied a reported ROS-generating effector of insulin and insulin-like growth factor signaling, NADPH oxidase 4 (Nox4). It is thought that Nox4 is both upregulated by insulin signaling and synergizes with it downstream via ROS-induced sustained phosphorylation of Akt. Our work in CGNPs revealed a marked induction of Nox4 in response to Shh at the mRNA and protein levels as well as an increase in total reactive oxygen species content. Subsequent studies suggest that Nox4 activity is critical to sustaining proliferation of Shh driven CGNPs. Western blots of shRNA knockdowns of Nox4 showed reduction of proliferative marker CyclinD2. The knockdowns also precipitated a drop in phosphorylated Akt perhaps leaving the Shh pathway without the full effects of one of its major signaling partners, the PI3K/Akt pathway.

MB-02 * SONIC HEDGEHOG INDUCES YB-1 IN A YAP-DEPENDENT MANNER TO REGULATE Igf2 EXPRESSION AND PROLIFERATION IN CEREBELLAR GRANULE NEURON PROGENITORS AND MEDULLOBLASTOMA CELLS

Interactions between the developmentally essential Sonic hedgehog (Shh) and Insulin-like Growth Factor (IGF) pathways play prominent roles in medulloblastoma (MB), the most common malignant pediatric brain tumor. MB patients undergo surgery, chemotherapy, and radiation, a regimen that carries devastating side effects, emphasizing the need for targeted therapies to improve survival and quality of life. Post-natal proliferation of cerebellar granule neuron precursors (CGNPs), proposed cells-of-origin for the SHH-associated subgroup of MB, is driven by Shh and IGF in the developing cerebellum. Shh induces the oncogene Yes-associated protein (YAP), which drives IGF2 expression in CGNPs and mouse Shh-associated medulloblastomas. To determine how IGF2 expression is regulated downstream of YAP, we carried out an unbiased screen for transcriptional regulators bound to IGF2 promoters. We report that Y-box binding protein-1 (YB-1), an onco-protein regulating transcription and translation, binds to IGF2 promoter P3 and is required for IGF2 expression in CGNPs. We show that YB-1 is induced by Shh in CGNPs in a YAP-dependent manner and is found in the germinal layer of the developing cerebellum. We observed that YB-1 is up-regulated across human medulloblastoma subclasses and is elevated in a mouse model for the Shh medulloblastoma subclass. Finally, shRNA-mediated knockdown experiments reveal that YB-1 activity is required for CGNP and medulloblastoma cell (MBC) proliferation in primary cultures and organotypic slice cultures. Collectively, our findings describe a novel role for YB-1 in driving proliferation in the developing cerebellum and medulloblastoma cells and they identify the SHH:YAP:YB1:IGF2 axis as a powerful target for therapeutic intervention in medulloblastomas.

MB-33 * THE CRITICAL ROLE OF OLIG2-EXPRESSING PROGENITORS IN THE INITIATION OF G-PROTEIN MEDIATED SONIC HEDGEHOG-DRIVEN MEDULLOBLASTOMA

Medulloblastoma, the most common malignant childhood brain tumor, exhibits distinct molecular subtypes and cellular origins. Genetic alterations driving medulloblastoma initiation and progression remain poorly understood. Recently we identify GNAS, encoding the G-protein Gsα, as a potent tumor suppressor gene that defines a subset of aggressive Sonic Hedgehog (Shh)-driven human medulloblastomas. Ablation of the single Gnas gene in anatomically-distinct progenitors is sufficient to induce Shh-associated medulloblastomas, which recapitulate their human counterparts. Gsα is highly enriched at the primary cilium of granule neuron precursors and suppresses Shh-signaling by regulating both the cAMP-dependent pathway and ciliary trafficking of Hedgehog pathway components. Elevation of a Gsα effector, cAMP, effectively inhibits tumor cell proliferation and progression in Gnas mutants. Furthermore, we identify a population of Olig2-expressing progenitor cells are the major source of tumor-propagating cells. Deletion of Olig2 leads to a delay of tumor progression. Eliminating the mitotic Olig2-expression inhibits tumor initiation. Thus, our present studies identify a previously unrecognized tumor suppressor function for Gsα that acts as a molecular link across Shh-group medulloblastomas of disparate cellular and anatomical origins, and reveal that Olig2-expressing progenitors are the critical cell origin of G-protein-mediated SHH subgroup medulloblastoma, illuminating potential therapeutic avenues for SHH medulloblastoma by targeting G-protein and Olig2-expressing glial progenitors.

The apical complex protein Pals1 is required to maintain cerebellar progenitor cells in a proliferative state.

Through their biased localization and function within the cell, polarity complex proteins are necessary to establish the cellular asymmetry required for tissue organization. Well-characterized germinal zones, mitogenic signals and cell types make the cerebellum an excellent model for addressing the crucial function of polarity complex proteins in the generation and organization of neural tissues. Deletion of the apical polarity complex protein Pals1 in the developing cerebellum results in a remarkably undersized cerebellum with disrupted layers in poorly formed folia and strikingly reduced granule cell production. We demonstrate that Pals1 is not only essential for cerebellum organogenesis, but also for preventing premature differentiation and thus maintaining progenitor pools in cerebellar germinal zones, including cerebellar granule neuron precursors in the external granule layer. In the Pals1 mouse mutants, the expression of genes that regulate the cell cycle was diminished, correlating with the loss of the proliferating cell population of germinal zones. Furthermore, enhanced Shh signaling through activated Smo cannot overcome impaired cerebellar cell generation, arguing for an epistatic role of Pals1 in proliferation capacity. Our study identifies Pals1 as a novel intrinsic factor that regulates the generation of cerebellar cells and Pals1 deficiency as a potential inhibitor of overactive mitogenic signaling.

Cross-species epigenetics identifies a critical role for VAV1 in SHH subgroup medulloblastoma maintenance.

The identification of key tumorigenic events in Sonic Hedgehog subgroup medulloblastomas (MBSHH) will be essential for the development of individualized therapies and improved outcomes. However, beyond confirmation of characteristic SHH-pathway mutations, recent genome-wide sequencing studies have not revealed commonly-mutated genes with widespread relevance as potential therapeutic targets. We therefore examined any role for epigenetic DNA methylation events in MBSHH using a cross-species approach to candidate identification, prioritization and validation. MBSHH–associated DNA methylation events were first identified in 216 subgrouped human medulloblastomas (50 MBSHH, 28 WNT, 44 Group 3, 94 Group 4) and their conservation then assessed in tumors arising from four independent murine models of Shh medulloblastoma, alongside any role in tumorigenesis using functional assessments in mouse and human models. This strategy identified widespread regional CpG hypo-methylation of VAV1, leading to its elevated expression, as a conserved aberrant epigenetic event which characterizes the majority of MBSHH tumors in both species, and is associated with a poor outcome in MBSHH patients. Moreover, direct modulation of VAV1 in mouse and human models revealed a critical role in tumor maintenance, and its abrogation markedly reduced medulloblastoma growth. Further, Vav1 activity regulated granule neuron precursor (GNP) germinal zone exit and migration initiation in an ex vivo model of early post-natal cerebellar development. These findings establish VAV1 as a critical epigenetically-regulated oncogene with a key role in MBSHH maintenance, and highlight its potential as a validated therapeutic target and prognostic biomarker for the improved therapy of medulloblastoma.

Endogenous cerebellar neurogenesis in adult mice with progressive ataxia.

ObjectiveTransplanting exogenous neuronal progenitors to replace damaged neurons in the adult brain following injury or neurodegenerative disorders and achieve functional amelioration is a realistic goal. However, studies so far have rarely taken into consideration the preexisting inflammation triggered by the disease process that could hamper the effectiveness of transplanted cells. Here, we examined the fate and long-term consequences of human cerebellar granule neuron precursors (GNP) transplanted into the cerebellum of Harlequin mice, an adult model of progressive cerebellar degeneration with early-onset microgliosis.MethodsHuman embryonic stem cell-derived progenitors expressing Atoh1, a transcription factor key to GNP specification, were generated in vitro and stereotaxically transplanted into the cerebellum of preataxic Harlequin mice. The histological and functional impact of these transplants was followed using immunolabeling and Rotarod analysis.ResultsAlthough transplanted GNPs did not survive beyond a few weeks, they triggered the proliferation of endogenous nestin-positive precursors in the leptomeninges that crossed the molecular layer and differentiated into mature neurons. These phenomena were accompanied by the preservation of the granule and Purkinje cell layers and delayed ataxic changes. In vitro neurosphere generation confirmed the enhanced neurogenic potential of the cerebellar leptomeninges of Harlequin mice transplanted with exogenous GNPs.InterpretationThe cerebellar leptomeninges of adult mice contain an endogenous neurogenic niche that can be stimulated to yield mature neurons from an as-yet unidentified population of progenitors. The transplantation of human GNPs not only stimulates this neurogenesis, but, despite the potentially hostile environment, leads to neuroprotection and functional amelioration.

Generation of Atoh1-rtTA transgenic mice: a tool for inducible gene expression in hair cells of the inner ear.

Atoh1 is a basic helix-loop-helix transcription factor that controls differentiation of hair cells (HCs) in the inner ear and its enhancer region has been used to create several HC-specific mouse lines. We generated a transgenic tetracycline-inducible mouse line (called Atoh1-rtTA) using the Atoh1 enhancer to drive expression of the reverse tetracycline transactivator (rtTA) protein and human placental alkaline phosphatase. Presence of the transgene was confirmed by alkaline phosphatase staining and rtTA activity was measured using two tetracycline operator (TetO) reporter alleles with doxycycline administered between postnatal days 0–3. This characterization of five founder lines demonstrated that Atoh1-rtTA is expressed in the majority of cochlear and utricular HCs. Although the tetracycline-inducible system is thought to produce transient changes in gene expression, reporter positive HCs were still observed at 6 weeks of age. To confirm that Atoh1-rtTA activity was specific to Atoh1-expressing cells, we also analyzed the cerebellum and found rtTA-driven reporter expression in cerebellar granule neuron precursor cells. The Atoh1-rtTA mouse line provides a powerful tool for the field and can be used in combination with other existing Cre recombinase mouse lines to manipulate expression of multiple genes at different times in the same animal.

Abstract B16: Dissecting MDM2 function in medulloblastoma pathogenesis

Abstract Treatment for medulloblastoma (MB), a tumor of the cerebellum, involves an aggressive regimen of surgical resection, radiation and adjuvant chemotherapy. Although the 5-year cure rate is over 50%, most children retain life-long side effects associated with treatment, including reduced intellect. There is an urgent need to develop multi-targeted therapeutic strategies that enhance the killing of MB tumor cells while sparing normal cells. As a critical component of the cellular response to stress, p53 function is required for the cytotoxic effects of many chemotherapeutics. As p53 mutations are relatively rare in MB, adjuvant chemotherapeutics that enhance p53 function may have a profound effect in MB. Indeed, we previously reported that pharmacological disruption of the interaction of p53 with its negative regulator, MDM2, potentiates cell killing in human medulloblastoma cells. In order to gain further insight into the role of MDM2 in MB pathogenesis, here we have used a combination of wild-type, null, and hypomorphic alleles of MDM2 to perturb the MDM2-p53 pathway in Ptch1+/- mice, a model of human Sonic hedgehog (Shh)-induced MB. Our studies demonstrate that a 70% reduction in the level of MDM2 abrogates the formation of preneoplastic lesions (PNLs) in the cerebellum of Ptch1+/- mice, thereby supporting a requirement for MDM2 in MB initiation. Significantly, even a modest ~50% reduction in the level of MDM2 was sufficient to decrease PNL formation in the Ptch1+/- cerebellum in two-fold. Although reduced in number, detailed histological analyses of PNLs at P21 revealed no difference in the size, proliferation or apoptosis of PNLs between Ptch1+/- mice expressing a wild-type or 50% reduced level of MDM2. Next, we examined granule neuron precursors (GNPs) in the external granule layer (EGL) at P7, the pool of cells from which PNLs are presumed to originate. Consistent with the partial deregulation of Shh signaling in Ptch1+/- mice, the Ptch1+/- EGL was more highly proliferative than that of wild-type mice. Importantly, we found that the enhanced proliferation of EGL cells afforded by loss of one Ptch1 allele was restored back to wild-type levels when the level of MDM2 is reduced. Additionally, a 50% in the level of MDM2 potentiates apoptosis and enhances the steady state level of p53SER15 in the EGL in Ptch1+/- mice as compared to control mice. Dysregulation of the MDM2-p53 and Sonic hedgehog (Shh)-Gli pathways are both implicated in the pathogenesis of human MB; however, no approach to date has yielded a detailed understanding of how crosstalk between these two important pathways contributes to the proliferation, survival, and differentiation of GNPs, the presumed cell of origin for some MB subtypes. Our prior studies placed MDM2 at a critical node between the growth inhibiting the p53 pathway and the growth-promoting Shh-Gli pathway in normal cerebellar development. New work presented here further suggests that MDM2 is required for MB tumorigenesis to prevent p53 activation, which may in turn function to counteract deregulated Shh-mediated proliferation in the tumor initiating cell population. The coordinate regulation of these two important pathways may provide a mechanism to sustain tumor-promoting levels of Shh-Gli signaling without activating the tumor-suppressing functions of p53. By nature of its requirement in MB tumorigenesis, MDM2 may provide an important therapeutic target for boosting the efficacy of current MB treatments. Citation Format: Susan M. Mendrysa, Reem Malek. Dissecting MDM2 function in medulloblastoma pathogenesis. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B16.

Loss of Smarc Proteins Impairs Cerebellar Development

SMARCA4 (BRG1) and SMARCB1 (INI1) are tumor suppressor genes that are crucially involved in the formation of malignant rhabdoid tumors, such as atypical teratoid/rhabdoid tumor (AT/RT). AT/RTs typically affect infants and occur at various sites of the CNS with a particular frequency in the cerebellum. Here, granule neurons and their progenitors represent the most abundant cell type and are known to give rise to a subset of medulloblastoma, a histologically similar embryonal brain tumor. To test how Smarc proteins influence the development of granule neurons and whether this population may serve as cellular origin for AT/RTs, we specifically deleted Smarca4 and Smarcb1 in cerebellar granule cell precursors. Respective mutant mice displayed severe ataxia and motor coordination deficits, but did not develop any tumors. In fact, they suffered from a severely hypoplastic cerebellum due to a significant inhibition of granule neuron precursor proliferation. Molecularly, this was accompanied by an enhanced activity of Wnt/β-catenin signaling that, by itself, is known to cause a nearly identical phenotype. We further used an hGFAP-cre allele, which deleted Smarcb1 much earlier and in a wider neural precursor population, but we still did not detect any tumor formation in the CNS. In summary, our results emphasize cell-type-dependent roles of Smarc proteins and argue against cerebellar granule cells and other progeny of hGFAP-positive neural precursors as the cellular origin for AT/RTs.

Abstract 404: Function of Brg1 chromatin remodeling factor in sonic hedgehog-dependent medulloblastoma development

Abstract Medulloblastoma is the most common malignant pediatric brain tumor. Overactive Shh signaling in cerebellum granule neuron precursors (CGNPs) is the leading cause of the childhood medulloblastoma (Shh-subtype). Previously we have shown that chromatin remodeler Brg1 is required for Shh target gene expression, and Brg1 deletion reduced CGNP proliferation in developing cerebellum. Current study focuses on the function of Brg1 in mouse model of Shh-subtype medulloblastoma. In CGNP cultured from SmoM2 transgenic mice where Shh pathway is constitutively active, we found Brg1 is required for CGNP mitogenic target gene expression and proliferation. In SmoM2 medulloblastoma cultures, we observed that tumor cell growth was inhibited by conditional knockout of Brg1. In subcutaneous transplantation, we found that tumors were significantly shrunk upon induction of Brg1 deletion. Detailed analysis indicated that Shh-dependent mitogenic target genes decreased by loss of Brg1. Further evidences showed that medulloblastoma cell proliferation was significant inhibited by conditional knockout of Brg1. Effect of Brg1 on the chromatin environment of target genes during medulloblastoma development will be discussed. Our study will provide insights to the epigenetic mechanism of Shh-dependent tumor development and new therapeutic targets. Citation Format: Xuanming Shi, Zilai Zhang, Qiu Wang, Jiang Wu. Function of Brg1 chromatin remodeling factor in sonic hedgehog-dependent medulloblastoma development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 404. doi:10.1158/1538-7445.AM2014-404

Abstract 3978: Reactive oxygen species in Sonic hedgehog-driven proliferation of cerebellar granule neuron precursors

Abstract Medulloblastoma is the most common solid malignant pediatric brain tumor. These tumors arise in the cerebellum and can be molecularly subdivided into 4 consensus subgroups, one of which is marked by amplification and activation of Sonic hedgehog (Shh) pathway components and downstream targets. This subclass is proposed to arise from oncogenic transformation of cerebellar granule neuron precursors (CGNPs), whose expansion during post-natal brain development is driven by and requires activation of the Shh pathway. These tumors often demonstrate similarities with normal cerebellar development at the molecular level, thus allowing us to use primary CGNP cultures as a model system for the Sonic hedgehog (Shh) driven subclass of medulloblastoma. In addition to mitogens driving proliferation, it has been shown in the past that low levels of intracellular reactive oxygen species (ROS) are required for proliferation, through mechanisms as diverse as inhibition of receptor tyrosine phosphatases, stabilization of proliferation proteins, and modifications of metabolites, thus indicating additional roles for ROS in proliferation and perhaps tumor growth beyond their known capacity to cause DNA damage, thereby contributing to genomic instability and apoptosis. Although the Shh ligand does not bind to a receptor tyrosine kinase (RTK), it is known that Shh signaling cooperates with RTK- activated pathways such as the insulin-like growth factor pathway to drive proliferation. To determine whether intracellular ROS play a role in Shh-driven CGNP proliferation, we treated CGNPs with the ROS scavenger lipoic acid (LA) in the presence of Shh, and observed a significant decrease in proliferation. Conversely, addition of the ROS inducer tert-Butyl hydroperoxide to Shh treated CGNPs led to enhanced proliferation over Shh treatment alone. These results indicate that a certain level of ROS are required to support Shh-driven CGNP proliferation, and enhancing their levels can increase proliferation. To investigate whether Shh signaling may affect expression of ROS regulatory genes we carried out a qPCR analysis. We identified up-regulation of sod2, gstm1, and gsto1: genes known to respond to ROS and whose products neutralize ROS, over vehicle-treated CGNPs. Paradoxically, when we examined ROS regulatory enzyme expression in Shh-driven mouse medulloblastomas, we noted sharp drop in the expression of gstm1, gsto1, and sod2 compared to the adjacent cerebellum suggesting a reduced ability to inactivate ROS, which could be contributing to proliferation or DNA damage in this transgenic model. These findings suggest that normal Sonic hedgehog pathway activation contributes to the production and tight regulation of ROS via downstream effectors in addition to synergizing with ROS to drive proliferation, a highly regulated balance that may be lost in medulloblastoma. Citation Format: Chad R. Potts, Rachel D. Rotenberry, Anna M. Kenney. Reactive oxygen species in Sonic hedgehog-driven proliferation of cerebellar granule neuron precursors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3978. doi:10.1158/1538-7445.AM2014-3978

Isolation of distinct cell populations from the developing cerebellum by microdissection.

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. This method was first utilized in the study of Nestin-expressing progenitors (NEPs), a newly identified population of cells in the cerebellar external germinal layer (EGL). Using microdissection in combination with fluorescent-activated cell sorting (FACS), a pure population of NEPs was collected separately from conventional granule neuron precursors in the EGL and from other contaminating Nestin-expressing cells in the cerebellum. Without microdissection, functional analyses of NEPs would not have been possible with the current methods available, such as Percoll gradient centrifugation and laser capture microdissection. This technique can also be applied for use with various tissues that contain either recognizable regions or fluorescently-labeled cells. Most importantly, a major advantage of this microdissection technique is that isolated cells are living and can be cultured for further experimentation, which is currently not possible with other described methods.

The G protein α subunit Gαs is a tumor suppressor in Sonic hedgehog-driven medulloblastoma.

Medulloblastoma, the most common malignant childhood brain tumor, exhibits distinct molecular subtypes and cellular origins. Genetic alterations driving medulloblastoma initiation and progression remain poorly understood. Herein, we identify GNAS, encoding the G-protein Gsα, as a potent tumor suppressor gene that defines a subset of aggressive Sonic Hedgehog (Shh)-driven human medulloblastomas. Ablation of the single Gnas gene in anatomically-distinct progenitors is sufficient to induce Shh-associated medulloblastomas, which recapitulate their human counterparts. Gsα is highly enriched at the primary cilium of granule neuron precursors and suppresses Shh-signaling by regulating both the cAMP-dependent pathway and ciliary trafficking of Hedgehog pathway components. Elevation of a Gsα effector, cAMP, effectively inhibits tumor cell proliferation and progression in Gnas mutants. Thus, our gain- and loss-of-function studies identify a previously unrecognized tumor suppressor function for Gsα that acts as a molecular link across Shh-group medulloblastomas of disparate cellular and anatomical origins, illuminating G-protein modulation as a potential therapeutic avenue.

Genetic drivers of metastatic dissemination in sonic hedgehog medulloblastoma.

Leptomeningeal dissemination (LMD), the metastatic spread of tumor cells via the cerebrospinal fluid to the brain and spinal cord, is an ominous prognostic sign for patients with the pediatric brain tumor medulloblastoma. The need to reduce the risk of LMD has driven the development of aggressive treatment regimens, which cause disabling neurotoxic side effects in long-term survivors. Transposon-mediated mutagenesis studies in mice have revealed numerous candidate metastasis genes. Understanding how these genes drive LMD will require functional assessment using in vivo and cell culture models of medulloblastoma. We analyzed two genes that were sites of frequent transposon insertion and highly expressed in human medulloblastomas: Arnt (aryl hydrocarbon receptor nuclear translocator) and Gdi2 (GDP dissociation inhibitor 2). Here we show that ectopic expression of Arnt and Gdi2 promoted LMD in mice bearing Sonic hedgehog (Shh)-induced medulloblastomas. We overexpressed Arnt and Gdi2 in a human medulloblastoma cell line (DAOY) and an immortalized, nontransformed cell line derived from mouse granule neuron precursors (SHH-NPD) and quantified migration, invasiveness, and anchorage-independent growth, cell traits that are associated with metastatic competence in carcinomas. In SHH-NPD cells. Arnt and Gdi2 stimulated all three traits. In DAOY cells, Arnt had the same effects, but Gdi2 stimulated invasiveness only. These results support a mechanism whereby Arnt and Gdi2 cause cells to detach from the primary tumor mass by increasing cell motility and invasiveness. By conferring to tumor cells the ability to proliferate without surface attachment, Arnt and Gdi2 favor the formation of stable colonies of cells capable of seeding the leptomeninges.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-014-0085-y) contains supplementary material, which is available to authorized users.