Development Of Sympathetic Nervous System Research Articles

Motor innervation directs the correct development of the mouse sympathetic nervous system

The sympathetic nervous system controls bodily functions including vascular tone, cardiac rhythm, and the “fight-or-flight response”. Sympathetic chain ganglia develop in parallel with preganglionic motor nerves extending from the neural tube, raising the question of whether axon targeting contributes to sympathetic chain formation. Using nerve-selective genetic ablations and lineage tracing in mouse, we reveal that motor nerve-associated Schwann cell precursors (SCPs) contribute sympathetic neurons and satellite glia after the initial seeding of sympathetic ganglia by neural crest. Motor nerve ablation causes mispositioning of SCP-derived sympathoblasts as well as sympathetic chain hypoplasia and fragmentation. Sympathetic neurons in motor-ablated embryos project precociously and abnormally towards dorsal root ganglia, eventually resulting in fusion of sympathetic and sensory ganglia. Cell interaction analysis identifies semaphorins as potential motor nerve-derived signaling molecules regulating sympathoblast positioning and outgrowth. Overall, central innervation functions both as infrastructure and regulatory niche to ensure the integrity of peripheral ganglia morphogenesis.

TMOD-27. IDENTIFYING ONCOGENIC C-MYC AND MYCN COMPLEXES IN HIGH-RISK PEDIATRIC CANCERS

Abstract The MYC family of proto-oncogenes is activated in a variety of cancers, including multiple high-risk pediatric malignancies. c-MYC (MYC) is ubiquitously expressed in human tissues, while MYCN (MYCN) has tissue and developmentally restricted expression patterns. In both neuroblastoma and medulloblastoma, enhanced activity of either MYCN or c-MYC drives high-risk disease. As transcription factors, MYC proteins exert oncogenic functions through protein-protein interaction networks that alter gene expression, but also mediate a growing list of target-gene independent nuclear functions (transcriptional elongation, chromatin changes throughout the cell cycle, etc…). While c-MYC and MYCN share many functions, they also regulate distinct cellular processes, and within medulloblastoma, they are activated in distinct molecular sub-groups (i.e. MYCN amplification is found in aggressive sonic hedge hog (SHH) subgroup tumors, while MYC amplification is found in aggressive group 3 and group 4 tumors). Here, we present an approach to identify oncogenic functions of c-MYC and MYCN in medulloblastoma and neuroblastoma using human induced pluripotent stem cell (iPSCO based orthotopic model systems. We hypothesize that the protein interaction networks and oncogenic functions of c-MYC and MYCN are impacted by cellular context, which are recapitulated in our orthotopic models (cell transcriptional and epigenetic landscape, tumor microenvironment). This premise is supported by recent single cell sequencing efforts in medulloblastoma and neuroblastoma, where primary human tumor cells are found to recapitulate specific transcriptional cell states found in normal hindbrain and sympathetic nervous system development, respectively. Through proximity labeling and quantitative mass spectrometry, we aim to identify tumor and oncogene specific protein interaction networks. This information will guide functional screening approaches to identify tumor-specific vulnerabilities. * Note MYC(N) refers to c-MYC and MYCN.

The sympathetic nervous system in development and disease.

The sympathetic nervous system prepares the body for 'fight or flight' responses and maintains homeostasis during daily activities such as exercise, eating a meal or regulation of body temperature. Sympathetic regulation of bodily functions requires the establishment and refinement of anatomically and functionally precise connections between postganglionic sympathetic neurons and peripheral organs distributed widely throughout the body. Mechanistic studies of key events in the formation of postganglionic sympathetic neurons during embryonic and early postnatal life, including axon growth, target innervation, neuron survival, and dendrite growth and synapse formation, have advanced the understanding of how neuronal development is shaped by interactions with peripheral tissues and organs. Recent progress has also been made in identifying how the cellular and molecular diversity of sympathetic neurons is established to meet the functional demands of peripheral organs. In this Review, we summarize current knowledge of signalling pathways underlying the development of the sympathetic nervous system. These findings have implications for unravelling the contribution of sympathetic dysfunction stemming, in part, from developmental perturbations to the pathophysiology of peripheral neuropathies and cardiovascular and metabolic disorders.

The embryonic trunk neural crest microenvironment regulates the plasticity and invasion of human neuroblastoma via TrkB signaling

Mistakes in trunk neural crest (NC) cell migration may lead to birth defects of the sympathetic nervous system (SNS) and neuroblastoma (NB) cancer. Receptor tyrosine kinase B (TrkB) and its ligand BDNF critically regulate NC cell migration during normal SNS development and elevated expression of TrkB is correlated with high-risk NB patients. However, in the absence of a model with in vivo interrogation of human NB cell and gene expression dynamics, the mechanistic role of TrkB in NB disease progression remains unclear. Here, we study the functional relationship between TrkB, cell invasion and plasticity of human NB cells by taking advantage of our validated in vivo chick embryo transplant model. We find that LAN5 (high TrkB) and SHSY5Y (moderate TrkB) human NB cells aggressively invade host embryos and populate typical NC targets, however loss of TrkB function significantly reduces cell invasion. In contrast, NB1643 (low TrkB) cells remain near the transplant site, but over-expression of TrkB leads to significant cell invasion. Invasive NB cells show enhanced expression of genes indicative of the most invasive host NC cells. In contrast, transplanted human NB cells down-regulate known NB tumor initiating and stem cell markers. Human NB cells that remain within the dorsal neural tube transplant also show enhanced expression of cell differentiation genes, resulting in an improved disease outcome as predicted by a computational algorithm. These in vivo data support TrkB as an important biomarker and target to control NB aggressiveness and identify the chick embryonic trunk neural crest microenvironment as a source of signals to drive NB to a less aggressive state, likely acting at the dorsal neural tube.

Mouse mothers transfer metabolic mode

Microbiota Obesity and metabolic diseases tend to go together, and humans who become obese are also prone to type 2 diabetes and cardiovascular problems. Starting with the observation that offspring of germ-free mice tended to become obese on high-fat diets, Kimura et al. investigated how the presence of the microbiota might be protective in mice (see the Perspective by Ferguson). Short-chain fatty acids (SCFAs) from the microbiota are known to suppress insulin signaling and reduce fat deposition in adipocytes. Further experiments showed that SCFAs in the bloodstream were able to pass from a non–germ-free mother's gut microbiota across the placenta and into the developing embryos. The authors found that in the embryos, the SCFA propionate mediates not only insulin levels through GPR43 signaling but also sympathetic nervous system development through GPR41 signaling. A high-fiber diet promoted propionate production from the maternal microbiota, and maternal antibiotic treatment resulted in obese-prone offspring. Science , this issue p. [eaaw8429][1]; see also p. [978][2] [1]: /lookup/doi/10.1126/science.aaw8429 [2]: /lookup/doi/10.1126/science.aba7673

Maternal gut microbiota in pregnancy influences offspring metabolic phenotype in mice

Antibiotics and dietary habits can affect the gut microbial community, thus influencing disease susceptibility. Although the effect of microbiota on the postnatal environment has been well documented, much less is known regarding the impact of gut microbiota at the embryonic stage. Here we show that maternal microbiota shapes the metabolic system of offspring in mice. During pregnancy, short-chain fatty acids produced by the maternal microbiota dictate the differentiation of neural, intestinal, and pancreatic cells through embryonic GPR41 and GPR43. This developmental process helps maintain postnatal energy homeostasis, as evidenced by the fact that offspring from germ-free mothers are highly susceptible to metabolic syndrome, even when reared under conventional conditions. Thus, our findings elaborate on a link between the maternal gut environment and the developmental origin of metabolic syndrome.

Differences in Genomic Profiles and Outcomes Between Thoracic and Adrenal Neuroblastoma

Neuroblastoma is a biologically and clinically heterogeneous disease. Based on recent studies demonstrating an association between the primary tumor site, prognosis, and commonly measured tumor biological features, we hypothesized that neuroblastomas arising in different sites would show distinct genomic features reflective of the developmental biology of the sympathicoadrenal nervous system. We first compared genomic and epigenomic data of primary diagnostic neuroblastomas originating in the adrenal gland (n = 646) compared to thoracic sympathetic ganglia (n = 118). We also evaluated association of common germline variation with these primary sites in 1027 European-American neuroblastoma patients. We observed higher rates of MYCN amplification, chromosome 1q gain, and chromosome 11q deletion among adrenal tumors, which were highly predictive of functional RNA signatures. Surprisingly, thoracic neuroblastomas were more likely to harbor ALK driver mutations than adrenal cases among all cases (odds ratio = 1.89, 95% confidence interval = 1.04 to 3.43), and among cases without MYCN amplification (odds ratio = 2.86, 95% confidence interval = 1.48 to 5.49). Common germline single nucleotide polymorphisms (SNPs) in BARD1 (previously associated with high-risk neuroblastoma) were found to be strongly associated with predisposition for origin at adrenal, rather than thoracic, sites. Neuroblastomas arising in the adrenal gland are more likely to harbor structural DNA aberrations including MYCN amplification, whereas thoracic tumors show defects in mitotic checkpoints resulting in hyperdiploidy. Despite the general association of ALK mutations with high-risk disease, thoracic tumors are more likely to harbor gain-of-function ALK aberrations. Site of origin is likely reflective of stage of sympathetic nervous system development when malignant transformation occurs and is a surrogate for underlying tumor biology.

Genomic Landscape of Multiple Myeloma with Elevated Lactate Dehydrogenase

BackgroundMultiple Myeloma (MM) is a heterogeneous disorder of clonal plasma cells. Genetic aberrations are important in heterogeneity and have prognostic and perhaps therapeutic implications. Elevated Lactate Dehydrogenase (LDH) has been associated with drug resistance and short survival. The biologic basis of this observation is uncertain. In this study, we sought to define the genomic landscape of MM patients with high LDH to understand pathophysiology and identify therapeutic targets.MethodsUtilizing data from the Multiple Myeloma Research Foundation (MMRF) CoMMpass database (IA12), which includes over 1000 newly-diagnosed MM patients with enriched tumor and matched constitutional samples analyzed using whole genome/exome and RNA sequencing (RNA-seq), we identified a cohort of patients with baseline LDH values and RNA-seq data available for inclusion. High LDH was defined as LDH greater than upper limit of normal (>4.68 microkatals/L). The RNA-seq data was analyzed to predict differentially expressed genes, then gene set enrichment analyses using GSEA and ClueGO were performed to assess for highly enriched pathways and gene ontologies (GO). Thereafter we analyzed to see if there was an enrichment of high risk cytogenetic changes within the high LDH group. Overall survival (OS) was estimated by Kaplan Meier method and a log-rank test.ResultsWe identified 871 patients who met inclusion criteria (High LDH N=143; Normal LDH N=728). LDH continued to remain a poor prognostic factor consistent with prior literature, with median survival 660 days vs 795 days (p=0.02852).Among the patients who underwent autologous transplant (N=385), LDH continued to be associated with poor prognosis with median overall survival (800.5 vs 878.8 days, p=0.01933). Patient characteristics and other clinical variables are submitted separately (Bal et al. ASH 2018).There was no difference in the non-synchronous mutations between the two groups when stratified by baseline LDH levels.To assess for enrichment of the known cytogenetic changes, we performed the hypergeometric test on the samples with both baseline LDH and cytogenetic information. Del(17p13) was significantly enriched (p=0.011) in the high LDH subset compared to normal LDH. (18.48% vs 10.36%) while there was no statistically significant difference in the presence of t(4;14) (p=0.16, 15.65% vs 11.8%) and t(14;16) (p=0.21, 6% vs 4%).GSEA detected 572 gene sets significantly up-regulated in the high LDH group (FDR q < 25%) compared to those with normal LDH including genes involved in the processing of capped intron containing pre-mRNA, recruitment of mitotic centrosome proteins and complexes, mRNA splicing and the proliferation signal in solid tumors leading to metastatic potential. No significantly down-regulated gene set was detected at same significance level.The ClueGO analysis using two separate sets of up-regulated DEGs (fold > 1.5x or fold > 2x, FDR < 0.05 for both) revealed upregulated molecular signatures in similar functional categories as the GSEA in patients with high LDH. The first gene set (fold > 1.5x) showed significant enrichments (p < 0.005) in cell cycle-related pathways, including microtubule cytoskeleton organization, polo-like kinase mediated events, regulation of cell cycle phase transition, regulation of nuclear division and kinesins which provide the myeloma cells with a proliferative advantage. The second gene set (fold > 2x) was strongly associated (p < 0.005) with sympathetic nervous system development (NELL2, NTRK1, and SOX11), collagen biosynthesis (ADAMTS family) and O-linked glycosylation (COL11A family). These genes play a role in lymphocyte differentiation, anti-apoptosis, local invasion, and metastasis.ConclusionElevated LDH was confirmed as a poor prognostic factor in the MMRF CoMMpass cohort. Overrepresentation of Del17p in this population likely contributes to poor prognosis. In MM, the bone marrow microenvironment is crucial in the differentiation, migration, proliferation and survival. Overexpression of proteolytic and cell adhesion signatures, evasion/suppression of host immune system along with hyper-proliferative signatures via cell division and RTK pathways in MM patients with high LDH offers insight into the aggressive disease in these patients. Targeting tumor microenvironment and RTK pathways may provide novel therapeutic strategies in this subtype of MM DisclosuresNo relevant conflicts of interest to declare.

The deubiquitinating enzyme UCHL1 is a favorable prognostic marker in neuroblastoma as it promotes neuronal differentiation

BackgroundNeuroblastoma (NB) is the most common pediatric solid tumor that originates from neural crest-derived sympathoadrenal precursor cells that are committed to development of sympathetic nervous system. The well differentiated histological phenotype of NB tumor cells has been reportedly associated with favorable patient outcome. Retinoic acid (RA) can effectively induce NB cell differentiation, thereby being used in the clinic as a treatment agent for inducing the differentiation of high-risk NB. However, the underlying molecular mechanisms of regulating differentiation remain elusive.MethodsThe correlation between clinical characteristics, survival and the deubiquitinating enzyme ubiquitin C-terminal hydrolase 1 (UCHL1) expression were assessed using a neuroblastic tumor tissue microarray, and then validated in three independent patient datasets. The different expression of UCHL1 in ganglioneuroblastoma, ganglioneuroma and NB was detected by immunohistochemistry, mass spectra and immunoblotting analysis, and the correlation between UCHL1 expression and the differentiated histology was analyzed, which was also validated in three independent patient datasets. Furthermore, the roles of UCHL1 in NB cell differentiation and proliferation and the underlying mechanisms were studied by using short hairpin RNA and its inhibitor LDN57444 in vitro.ResultsBased on our neuroblastic tumor tissue microarrays and three independent validation datasets (Oberthuer, Versteeg and Seeger), we identified that UCHL1 served as a prognostic marker for better clinical outcome in NB. We further demonstrated that high UCHL1 expression was associated with NB differentiation, indicated by higher UCHL1 expression in ganglioneuroblastomas/ganglioneuromas and well-differentiated NB than poorly differentiated NB, and the positive correlation between UCHL1 and differentiation markers. As expected, inhibiting UCHL1 by knockdown or LDN57444 could significantly inhibit RA-induced neural differentiation of NB tumor cells, characterized by decreased neurite outgrowth and neural differentiation markers. This effect of UCHL1 was associated with positively regulating RA-induced AKT and ERK1/2 signaling activation. What’s more, knockdown of UCHL1 conferred resistance to RA-induced growth arrest.ConclusionOur findings identify a pivotal role of UCHL1 in NB cell differentiation and as a prognostic marker for survival in patients with NB, potentially providing a novel therapeutic target for NB.

Temporal requirements for ISL1 in sympathetic neuron proliferation, differentiation, and diversification

Malformations of the sympathetic nervous system have been associated with cardiovascular instability, gastrointestinal dysfunction, and neuroblastoma. A better understanding of the factors regulating sympathetic nervous system development is critical to the development of potential therapies. Here, we have uncovered a temporal requirement for the LIM homeodomain transcription factor ISL1 during sympathetic nervous system development by the analysis of two mutant mouse lines: an Isl1 hypomorphic line and mice with Isl1 ablated in neural crest lineages. During early development, ISL1 is required for sympathetic neuronal fate determination, differentiation, and repression of glial differentiation, although it is dispensable for initial noradrenergic differentiation. ISL1 also plays an essential role in sympathetic neuron proliferation by controlling cell cycle gene expression. During later development, ISL1 is required for axon growth and sympathetic neuron diversification by maintaining noradrenergic differentiation, but repressing cholinergic differentiation. RNA-seq analyses of sympathetic ganglia from Isl1 mutant and control embryos, together with ISL1 ChIP-seq analysis on sympathetic ganglia, demonstrated that ISL1 regulates directly or indirectly several distinct signaling pathways that orchestrate sympathetic neurogenesis. A number of genes implicated in neuroblastoma pathogenesis are direct downstream targets of ISL1. Our study revealed a temporal requirement for ISL1 in multiple aspects of sympathetic neuron development, and suggested Isl1 as a candidate gene for neuroblastoma.

From proliferation to target innervation: signaling molecules that direct sympathetic nervous system development

The sympathetic division of the autonomic nervous system includes a variety of cells including neurons, endocrine cells and glial cells. A recent study (Furlan et al. 2017) has revised thinking about the developmental origin of these cells. It now appears that sympathetic neurons and chromaffin cells of the adrenal medulla do not have an immediate common ancestor in the form a "sympathoadrenal cell", as has been long believed. Instead, chromaffin cells arise from Schwann cell precursors. This review integrates the new findings with the expanding body of knowledge on the signalling pathways and transcription factors that regulate the origin of cells of the sympathetic division of the autonomic nervous system.

Trajectories of sleep and cardiac sympathetic activity indexed by pre-ejection period in childhood.

Fragmented and insufficient sleep has been implicated in disrupted autonomic nervous system activity during resting state conditions in typically developing children. Towards explication of these relations over development, the current study tested reciprocal relations between the development of sleep parameters (efficiency, duration, latency) and cardiac sympathetic nervous system (SNS) activity indexed by pre-ejection period (PEP) during waking-resting state conditions throughout middle and late childhood. Whether sleep derives changes in PEP or vice versa was examined. A longitudinal design was employed and latent growth modelling was used to examine the research questions. During the first assessment, 282 children aged 9.44years (65% European American, 35% African American) participated. Two more assessments followed, with a 1-year lag between consecutive study waves. Sleep was examined with 7 nights of actigraphy in the child's home. Controlling for many potential confounds (sex, race/ethnicity, body mass index and family socioeconomic status), higher sleep efficiency and more sleep minutes predicted increases in PEP (less SNS activity) over 3years. PEP did not predict changes in sleep efficiency or duration over time and there were no significant effects for sleep latency. Findings highlight the probable direction of effects between these two key bioregulatory systems. High levels of cardiac SNS activity are associated with many negative health outcomes, and thus these findings may have important implications.

Emergent Role of Coronin-1a in Neuronal Signaling.

The Coronin family of proteins were first noted for their role in pathogen-host interactions and for modulating actin dynamics. Recently, however, Coronins have been found in a greater variety of cell types, and novel roles for the Coronins within the nervous system have been discovered. In the immune system, Coronin-1a enables Mycobacterium tuberculosis to evade lysosomal destruction. This activity appears to be analogous to protection of the NGF-TrkA signaling endosome during sympathetic nervous system development that is required for survival signaling. Similarly, others have implicated Coronin-1a in GPCR signaling during the formation of excitatory connections in the central nervous system. Its role in multiple signaling pathways suggests that it may influence cross talk between key pathways (TrkA, GPCRs) during neurodevelopment. Here, we review the role of Coronin-1a in neural development and function.

Abstract 2456: Synergy between loss of NF1 and overexpression of MYCN in neuroblastoma is mediated by the GAP-related domain

Abstract Earlier reports indicated that the role of Nf1 tumor suppressor gene in limiting sympathoadrenal cell growth during embryologic development is independent of its ability to down-modulate RAS-MAPK signaling. This finding raised the question of whether neuroblastoma pathogenesis was also accelerated by loss of a similar non-canonical function of NF1. To elucidate how loss of the NF1 tumor suppressor gene contributes to the development of high-risk neuroblastoma, we relied on a transgenic zebrafish model that overexpresses MYCN and harbors loss-of-function nf1 mutations. We show here that loss of nf1 leads to aberrant activation of RAS signaling in MYCN-induced neuroblastoma, promoting both increased tumor cell survival and rapid tumor cell proliferation. We demonstrate further that the GTPase-activating protein (GAP) activity of the (GAP)-related domain (GRD) is sufficient to suppress accelerated initiation of neuroblastoma in nf1-deficient zebrafish, even though this transgene is unable to restrict abnormal sympathoadrenal cell growth during embryologic development. Hence NF1 exhibits different activities in vivo in the normal development and tumorigenesis of the peripheral sympathetic nervous system. Our findings establish nf1-deficient zebrafish that overexpress MYCN as an ideal animal model system for investigating new strategies to improve treatment of very high risk neuroblastomas with aberrant RAS-MAPK activation. We are currently performing high-throughput in vivo drug analysis using these zebrafish with primary tumors. Citation Format: Shuning He, Marc R. Mansour, Mark W. Zimmerman, Dong Hyuk Ki, Hillary M. Layden, Koshi Akahane, Eric D. de Groh, Antonio R. Perez-Atayde, Shizhen Zhu, Jonathan A. Epstein, A Thomas Look. Synergy between loss of NF1 and overexpression of MYCN in neuroblastoma is mediated by the GAP-related domain. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2456.

Abstract A22: Differentiation of human embryonic stem cells to sympathetic neurons: A potential model for understanding neuroblastoma pathogenesis

Abstract Background and Aims: Neuroblastoma is an embryonal malignancy derived from neural crest cells which give rise to the sympathetic nervous system (SNS). One of the main therapeutic challenges in neuroblastoma continues to be the emergence of relapsed disease, which occurs in 50% of patients with high risk disease and is associated with resistance to therapy. Our aim was to differentiate human embryonic stem cells (hESCs) to sympathetic neurons (SN) to model normal human SNS development. Results: Using the stromal-derived inducing activity (SDIA) of murine PA6 cells in combination with BMP4 and B27 neuronal supplement, H9 and a newly derived hESC line Ncl(R)14, were induced to differentiate to neural crest stem cells and SN. After 7 days of PA6 cell co-culture, mRNA expression of SNAIL and SOX-9 neural crest specifier genes and the neural marker Peripherin increased. Q-RT-PCR showed that expression of the pluripotency marker OCT 4 decreased, whereas p53 and LIN28B expression remained high at levels similar to SHSY5Y and IMR32 neuroblastoma cell lines. A marked increase in expression of the catecholaminergic marker Tyrosine Hydroxylase (TH) and the noradrenergic marker Dopamine Beta Hydroxylase (DBH) was observed by day 7 of differentiation. Fluorescence activated cell sorting for the neural crest marker p75, enriched for cells expressing p75, DBH, TH and Peripherin. SN were identified by immunofluorescence by co-expression of TH &amp; Peripherin or DBH &amp; PHOX2B in p75+ cells. Live cell analysis and imaging showed increased migration in p75+ cells compared with p75- cells, consistent with a migratory neural crest phenotype in-vitro. Conclusions: We have established a model of nor-adrenergic SNS development using two hESC lines to improve our understanding of normal human SNS development and in future studies the pathogenesis of neuroblastoma. Citation Format: Jane Carr-Wilkinson, Nilendran Prathalingam, Deepali Pal, Helen Forgham, Majlinda Lako, Mary Herbert, Deborah A. Tweddle. Differentiation of human embryonic stem cells to sympathetic neurons: A potential model for understanding neuroblastoma pathogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A22.

Studying the peripheral sympathetic nervous system and neuroblastoma in zebrafish.

The zebrafish serves as an excellent model to study vertebrate development and disease. Optically clear embryos, combined with tissue-specific fluorescent reporters, permitdirect visualization and measurement of peripheral nervous system formation in real time. Additionally, the model is amenable to rapid cellular, molecular, and genetic approaches to determine how developmental mechanisms contribute to disease states, such as cancer. In this chapter, we describe the development of the peripheral sympathetic nervous system (PSNS) in general, and our current understanding of genetic pathways important in zebrafish PSNS development specifically. We also illustrate how zebrafish genetics is used to identify new mechanisms controlling PSNS development and methods for interrogating the potential role of PSNS developmental pathways in neuroblastoma pathogenesis in vivo using the zebrafish MYCN-driven neuroblastoma model.

Transcription factor activating protein 2 beta (TFAP2B) mediates noradrenergic neuronal differentiation in neuroblastoma

Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2′-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma.

TNFR family members cooperate to mediate sympathetic nervous system development

TNFR family members cooperate to mediate sympathetic nervous system development

Cyclin D1 in pediatric neuroblastic tumors: A microarray analysis

Neuroblastoma is the most common extracranial solid childhood tumor, which is believed to originate from primitive neuroblasts giving rise to the sympathetic nervous system. It was previously shown that cyclin D1 (CCDN1) in pediatric neuroblastic tumors (neuroblastoma, ganglioneuroblastoma, and ganglioneuroma) recapitulates its expression during the development of peripheral sympathetic nervous system (PSNS). In the present study, we performed a microarray analysis in order to evaluate the expression of cyclin D1 in neuroblastoma as compared to ganglioneuroma and ganglioneuroblastoma. We first confirmed that comparable levels of cyclin D1 are present in neuroblastoma and fetal neuroblasts. In addition, we observed that neuroblastoma is associated to significantly higher levels of cyclin D1 as compared to both ganglioneuroma and ganglioneuroblastoma. No differences are instead observable between ganglioneuroblastoma and ganglioneuroma. Finally, bioinformatic analysis of cyclin D1-functionally related genes, identified cyclin D2 as an additional marker/etiopathogenic factor in the development of neuroblastoma.

Abstract A04: Metabolic reprogramming of MYCN amplified neuroblastoma

Abstract Neuroblastoma (NB) is a highly malignant embryonic tumor of neural crest origin and accounts for 15% of all cancer deaths in children. Amplification of the MYCN oncogene, which occurs in ~25% of patients, is the most reliable marker of metastatic disease and predicts poor outcome. Recent findings show MYCN reprograms NB cell metabolism to promote glycolysis and shunting of glutamate into the TCA cycle, causing dependence on increased levels of TCA intermediates, such as a-KG and succinate. Allelic loss of the short arm of chromosome 1, (1p36) is associated with the vast majority of MYCN amplified NB, and a candidate tumor suppressor gene (TSG) in this region is succinate dehydrogenase subunit B,SDHB, which encodes a core component of the succinate dehydrogenase (SDH) complex, converting succinate to fumarate. We show that MYCN-amplified NB cells, with 1p deletion, have reduced SDHB protein levels and elevated succinate levels. Thus, we are now testing a model that the combined loss of SDH activity with MYCN amplification causes dramatic accumulation of cytosolic succinate, which in turn stabilizes HIF1α to accelerate NB pathogenesis in vivo. To test this model, we have generated mutations in zebrafish sdhb to determine if 1) it is normally required for sympathetic neuron differentiation and 2) cooperates with MYCN to accelerate zebrafish NB in vivo (Zhu et. al, Cancer Cell, 2012). As HIF1a is predicted to be upregulated in SDH-deficient cells, as well as MYCN-amplified NB, we have also generated stable HIF1α transgenic zebrafish lines to directly determine if HIF1α alone is sufficient to drive NB or cooperates with MYCN to accelerate it. The results from our studies of sdhb loss and hif1α gain on peripheral sympathetic nervous system development and NB formation in vivo will be discussed as well as potential therapeutic approaches to target SDH/MYCN-dependent tumors. Citation Format: Monique Morrison, Scott Khuu, Joshua Schiffman, Bradley Cairns, Jared Rutter, Dennis Winge, Frank Speleman, Rodney Stewart. Metabolic reprogramming of MYCN amplified neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A04.