Target In Neuroblastoma Research Articles

Cholesterol Biosynthesis Is Required for Cell Death in Response to G9A Inhibitor in Lung Cancer (P05-010-19)

ObjectivesG9A is a eukaryotic histone methyltransferase that contributes to transcriptional silencing of tumor suppressor genes by modulating histone 3 lysine 9 methylation (H3K9m). It has been recently suggested as a promising therapeutic target for breast cancer and neuroblastoma. This study was aimed to investigate the effect of G9A inhibition and its cellular metabolic mechanisms. MethodsUsing OncomineTM, G9A overexpression in lung cancer was assessed. Cell viability upon treatment of G9A inhibitor (BIX01294, BIX) and siG9A was measured by MTT and IncuCyteR assays. Additionally, apoptosis and autophagy were analyzed through western blots. In order to identify targets, transcriptomes using RNA sequencing was conducted upon BIX treatment. Further functional relevance of targets was validated using Chromatin IP and recovery tests. ResultsBIX-mediated inhibition of G9A reduced cell viability of lung cancer cells via induction of autophagy. Through RNA sequencing, we found that G9A inhibition mainly affected cholesterol biosynthesis pathway. BIX directly induced the expression of SREBF2 gene, by lowering H3K9me1 and H3K9me2 at the promoter. A cholesterol biosynthesis inhibitor, 25-HC, partially recovered BIX-induced cell death by attenuating autophagy. Our data suggests that cholesterol metabolism can be a potential therapeutic target by G9A inhibition and its induction of autophagic cell death. ConclusionsOur data suggests that cholesterol metabolism can be a potential therapeutic target by G9A inhibition and its induction of autophagic cell death. Funding SourcesNRF of Korea grant (2018R1D1A1B07051274); BK21 Plus Project (22A20130012143).

Vitronectin as a molecular player of the tumor microenvironment in neuroblastoma

BackgroundVitronectin is a multifunctional glycoprotein known in several human tumors for its adhesive role in processes such as cell growth, angiogenesis and metastasis. In this study, we examined vitronectin expression in neuroblastoma to investigate whether this molecule takes part in cell-cell or cell-extracellular matrix interactions that may confer mechanical properties to promote tumor aggressiveness.MethodsWe used immunohistochemistry and image analysis tools to characterize vitronectin expression and to test its prognostic value in 91 neuroblastoma patients. To better understand the effect of vitronectin, we studied its in vitro expression using commercial neuroblastoma cell lines and in vivo using intra-adrenal gland xenograft models by immunohistochemistry.ResultsDigital image analysis allowed us to associate vitronectin staining intensity and location discriminating between territorial vitronectin and interterritorial vitronectin expression patterns. High territorial vitronectin expression (strong staining associated with pericellular and intracellular location) was present in tumors from patients with metastatic undifferentiating neuroblastoma, that were MYCN amplified, 11q deleted or with segmental chromosomal profiles, in the high-risk stratification group and with high genetic instability. In vitro studies confirmed that vitronectin is expressed in tumor cells as small cytoplasmic dot drops. In vivo experiments revealed tumor cells with high and dense cytoplasmic vitronectin expression.ConclusionsThese findings highlight the relevance of vitronectin in neuroblastoma tumor biology and suggest its potential as a future therapeutic target in neuroblastoma.

Prohibitin promotes de-differentiation and is a potential therapeutic target in neuroblastoma.

Gain of the long arm of chromosome 17 (17q) is a cytogenetic hallmark of high-risk neuroblastoma, yet its contribution to neuroblastoma pathogenesis remains incompletely understood. Combining whole-genome and RNA sequencing of neuroblastomas, we identified the prohibitin (PHB) gene as highly expressed in tumors with 17q gain. High PHB expression correlated with poor prognosis and was associated with loss of gene expression programs promoting neuronal development and differentiation. PHB depletion induced differentiation and apoptosis and slowed cell cycle progression of neuroblastoma cells, at least in part through impaired ERK1/2 activation. Conversely, ectopic expression of PHB was sufficient to increase proliferation of neuroblastoma cells and was associated with suppression of markers associated with neuronal differentiation and favorable neuroblastoma outcome. Thus, PHB is a 17q oncogene in neuroblastoma that promotes tumor cell proliferation, and de-differentiation.

An antibody-drug conjugate directed to the ALK receptor demonstrates efficacy in preclinical models of neuroblastoma.

Enthusiasm for the use of antibody-drug conjugates (ADCs) in cancer therapy has risen over the past few years. The success of this therapeutic approach relies on the identification of cell surface antigens that are widely and selectively expressed on tumor cells. Studies have shown that native ALK protein is expressed on the surface of most neuroblastoma cells, providing an opportunity for development of immune-targeting strategies. Clinically relevant antibodies for this target have not yet been developed. Here, we describe the development of an ALK-ADC, CDX-0125-TEI, which selectively targets both wild-type and mutated ALK-expressing neuroblastomas. CDX-0125-TEI exhibited efficient antigen binding and internalization, and cytotoxicity at picomolar concentrations in cells with different expression of ALK on the cell surface. In vivo studies showed that CDX-0125-TEI is effective against ALK wild-type and mutant patient-derived xenograft models. These data demonstrate that ALK is a bona fide immunotherapeutic target and provide a rationale for clinical development of an ALK-ADC approach for neuroblastomas and other ALK-expressing childhood cancers such as rhabdomyosarcomas.

Dual-Specificity Phosphatases in Neuroblastoma Cell Growth and Differentiation.

Dual-specificity phosphatases (DUSPs) are important regulators of neuronal cell growth and differentiation by targeting proteins essential to neuronal survival in signaling pathways, among which the MAP kinases (MAPKs) stand out. DUSPs include the MAPK phosphatases (MKPs), a family of enzymes that directly dephosphorylate MAPKs, as well as the small-size atypical DUSPs, a group of low molecular-weight enzymes which display more heterogeneous substrate specificity. Neuroblastoma (NB) is a malignancy intimately associated with the course of neuronal and neuroendocrine cell differentiation, and constitutes the source of more common extracranial solid pediatric tumors. Here, we review the current knowledge on the involvement of MKPs and small-size atypical DUSPs in NB cell growth and differentiation, and discuss the potential of DUSPs as predictive biomarkers and therapeutic targets in human NB.

Maternal Embryonic Leucine Zipper Kinase (MELK), a Potential Therapeutic Target for Neuroblastoma.

Maternal embryonic leucine zipper kinase (MELK) activates pathways that mediate aggressive tumor growth and therapy resistance in many types of adult cancers. Pharmacologic and genomic inhibition of MELK impairs tumor growth and increases sensitivity to radiation and chemotherapy. On the basis of these promising preclinical studies, early-phase adult clinical trials testing the MELK inhibitor OTS167 are ongoing. To investigate whether MELK is also a therapeutic target in neuroblastoma, we analyzed MELK expression in primary tumors and cell lines, and examined the effects of OTS167 on neuroblastoma growth. In primary tumors, high levels of MELK were associated with advanced stage disease and inferior survival. Higher levels of MELK were also detected in tumorigenic versus nontumorigenic neuroblastoma cell lines, and cells with higher levels of MELK expression were more sensitive to OTS167 than low-MELK expressing cells. OTS167 suppressed the growth of neuroblastoma xenografts, and in a preclinical model of minimal residual disease, survival was prolonged with MELK inhibition. OTS167 treatment downregulated MELK and its target enhancer of zeste homolog 2 (EZH2), a component of the polycomb repressive complex 2 (PRC2) that is known to modulate the DNA damage response. We also show that OTS167 reduced the formation of collapsed replication forks induced by camptothecin or radiation. Taken together, our results indicate that MELK indirectly mediates efficient processing of replication-associated DNA lesions in neuroblastoma, and that OTS167 sensitizes cells to DNA-damaging agents by abrogating this process. Further studies evaluating the activity of combination treatment regimens with OTS167 in neuroblastoma are warranted.

Highlights of This Issue

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and most patients have poor responses to conventional therapy. TGFβ signaling is required for evasion of the cytotoxic immune program in PDAC. However, pharmacologic inhibition of TGFβ signaling failed to promote an anti-tumor immune response in clinical trials. In murine PDAC, TGFβ signal inhibition similarly failed to alter disease course, and enhanced expression of the immune checkpoint molecule PD-L1. Concomitant TGFβ and PD-L1 pathway inhibition led to a significant reduction in the neoplastic phenotype, improving survival and reducing disease-associated morbidity in vivo. Hence, combined TGFβ/PD-L1 inhibition warrants therapeutic consideration in PDAC patients.High-risk neuroblastoma patients currently witness survival rates less than 50% as well as the potential for treatment-related late effects. There is therefore a need to identify exploitable targets in neuroblastoma that increase treatment efficacy and decrease toxicity. In this manuscript, Chlenski and colleagues investigated the Maternal Embryonic Leucine Zipper Kinase (MELK) as one such target. Inhibiting MELK with OTS167 significantly decreased the growth of neuroblastoma tumors in vivo. Furthermore, OTS167 prolonged the survival of mice whose tumors had previously achieved minimal residual disease via treatment with cyclophosphamide. OTS167 also synergized with the DNA damaging agent CPT and with radiation therapy. These results outline the potential efficacy of combining OTS167 with current therapies in neuroblastoma.Triple-negative breast cancer (TNBC) remains a difficult subtype due to the high rates of recurrence and metastatic spread. To combat this, Ramchandani, Lee, and colleagues have designed the first gold nanoparticle-delivered miRNA mimetic for TNBC. The nanoparticle utilizes a novel layer-by-layer construction that incorporates miR-708 and successfully delivers it to the primary tumor. By using a fluorescent reporter of the SOX2 and OCT4 response elements, they demonstrated miR-708 gold nanoparticles reduced the migration properties of invasive TNBC cell populations. Furthermore, administration of miR-708 nanoparticles in vivo reduced formation of metastatic nodules. Nanoparticles containing miR-708 therefore represent a potential anti-metastatic therapy for TNBC.Most patients with high-grade serous ovarian cancer (HGSOC) develop resistance to chemotherapy. In this work, Iavarone and colleagues demonstrated administration of the MEK inhibitor cobimetinib (GDC-0973) primed cells from patient-derived xenografts models for BCL-2-regulated apoptosis through the upregulation of BIM. Combining GDC-0973 with the BCL-2/XL inhibitor ABT-263 therefore led to significant reductions in cell number and was well-tolerated in vivo. Intuitively, combined therapy was efficacious in models for which BIM was either initially highly expressed or was induced with GDC-0973 treatment and was not efficacious in models where BIM was not induced. These data indicate a mechanistic rationale for the combined use of MEK and BCL-2/XL inhibitors in high-grade serous ovarian cancer.

SYK Inhibition Potentiates the Effect of Chemotherapeutic Drugs on Neuroblastoma Cells in Vitro.

Neuroblastoma is a malignancy arising from the developing sympathetic nervous system and the most common and deadly cancer of infancy. New therapies are needed to improve the prognosis for high-risk patients and to reduce toxicity and late effects. Spleen tyrosine kinase (SYK) has previously been identified as a promising drug target in various inflammatory diseases and cancers but has so far not been extensively studied as a potential therapeutic target in neuroblastoma. In this study, we observed elevated SYK gene expression in neuroblastoma compared to neural crest and benign neurofibroma. While SYK protein was detected in the majority of examined neuroblastoma tissues it was less frequently observed in neuroblastoma cell lines. Depletion of SYK by siRNA and the use of small molecule SYK inhibitors significantly reduced the cell viability of neuroblastoma cell lines expressing SYK protein. Moreover, SYK inhibition decreased ERK1/2 and Akt phosphorylation. The SYK inhibitor BAY 61-3606 enhanced the effect of different chemotherapeutic drugs. Transient expression of a constitutive active SYK variant increased the viability of neuroblastoma cells independent of endogenous SYK levels. Collectively, our findings suggest that targeting SYK in combination with conventional chemotherapy should be further evaluated as a treatment option in neuroblastoma.

SNW1 regulates Notch signaling in neuroblastoma through interacting with RBPJ

The Notch signaling pathway is highly conserved, involved in cell-cell communication, and essential for multicellular organism development. The recombination signal binding protein for immunoglobulin kappa J region (RBPJ) transcriptional regulator plays a vital role in Notch signaling. When not complexed with Notch proteins, RBPJ acts as a transcriptional repressor. However, when bound to the Notch intracellular domain (NOTCH ICD) in the nucleus, RBPJ activates the transcription of Notch target genes. However, the role of RBPJ in the Notch signaling pathway in neuroblastoma (NB) remains unclear. In this study, we show that SNW domain containing 1 (SNW1) acts as a novel RBPJ partner and regulates the expression of Notch target genes. Additionally, we show that silencing RBPJ expression can prevent SNW1-mediated Notch gene expression. Public datasets and clinical NB tissues indicate that RBPJ and SNW1 are upregulated in NB, and are associated with unfavorable patient outcomes. Taken together, our results highlight that SNW1 interacts with RBPJ to regulate the Notch signaling pathway and presents a potential therapeutic target for NB.

Covalent inhibition of histone deacetylase 8 by 3,4-dihydro-2H-pyrimido[1,2-c][1,3]benzothiazin-6-imine

BackgroundHDAC8 is an established target for T-cell lymphoma and childhood neuroblastoma. Benzothiazine-imines are promising HDAC8 inhibitors with unknown binding mechanism lacking a usual zinc binding group. MethodsIn this study high-resolution and quantitative HPLC-coupled ESI-MS/MS techniques are combined with crystal structure determination and a variety of biochemical and computational methods to elucidate the reaction mechanism between benzothiazine-imine 1 and HDAC8. Results1) 1 is a covalent inhibitor of HDAC8; 2) inhibition is reversible in the presence of reducing agents; 3) C153 in the active site and C102 are involved in the inhibition mechanism; 4) 1 modifies various cysteines in HDAC8 forming either thiocyanates or mixed disulfides with 3; 5) 1 and 5 dock in close proximity to C153 within the active site. This is supposed to accelerate covalent inactivation particularly in HDAC8 and suggested as major determinant for the observed nanomolar potency and selectivity of 1. Conclusions1 and its analogs are interesting model compounds but unsuitable for therapeutic treatment due to their high unselective reactivity towards thiol groups. However, the postulated preceding non-covalent binding mode of 1 opens a door to optimized next generation compounds that combine potent and selective non-covalent recognition with low reactivity towards C153 at the active site of HDAC8. General significance1 represents a completely new class of inhibitors for HDAC8. Initial non-covalent interaction at the bottom of the active site is suggested to be the key for its selectivity. Further optimization of non-covalent interaction and thiol-reactivity provides opportunities to develop therapeutic useful covalent HDAC8 inhibitors.

Collaborative ISL1/GATA3 interaction in controlling neuroblastoma oncogenic pathways overlapping with but distinct from MYCN.

Background: Transcription factor ISL1 plays a critical role in sympathetic neurogenesis. Expression of ISL1 has been associated with neuroblastoma, a pediatric tumor derived from sympatho-adrenal progenitors, however the role of ISL1 in neuroblastoma remains unexplored.Method: Here, we knocked down ISL1 (KD) in SH-SY5Y neuroblastoma cells and performed RNA-seq and ISL1 ChIP-seq analyses.Results: Analyses of these data revealed that ISL1 acts upstream of multiple oncogenic genes and pathways essential for neuroblastoma proliferation and differentiation, including LMO1 and LIN28B. ISL1 promotes expression of a number of cell cycle associated genes, but represses differentiation associated genes including RA receptors and the downstream target genes EPAS1 and CDKN1A. Consequently, Knockdown of ISL1 inhibits neuroblastoma cell proliferation and migration in vitro and impedes tumor growth in vivo, and enhances neuronal differentiation by RA treatment. Furthermore, genome-wide mapping revealed a substantial co-occupancy of binding regions by ISL1 and GATA3, and ISL1 physically interacts with GATA3, and together they synergistically regulate the aforementioned oncogenic pathways. In addition, analyses of the roles of ISL1 and MYCN in MYCN-amplified and MYCN non-amplified neuroblastoma cells revealed an epistatic relationship between ISL1 and MYCN. ISL1 and MYCN function in parallel to regulate common yet distinct oncogenic pathways in neuroblastoma.Conclusion: Our study has demonstrated that ISL1 plays an essential role in neuroblastoma regulatory networks and may serve as a potential therapeutic target in neuroblastoma.

Targeting cholesterol metabolism in neuroblastoma by acting on HDACs

Event Abstract Back to Event Targeting cholesterol metabolism in neuroblastoma by acting on HDACs Maria João Nunes1, 2, Daniela Costa1, Rita Mendes De Almeida1, Margarida Castro-Caldas1, 3, Maria João Gama1, 2 and Elsa Rodrigues1, 2* 1 Research Institute for Medicines (iMed.ULisboa), Portugal 2 Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Portugal 3 Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal A wide range of brain disorders are associated with imbalances in protein acetylation levels and treatment with various histone deacetylase (HDAC) inhibitors (HDACi) has been shown to correct these deficiencies, emerging as a promising new strategy for therapeutic intervention in neurodegenerative diseases. We have demonstrated that in neuroblastoma cells HDACi dramatically increase mRNA levels of genes involved in cholesterol efflux and elimination, while decreasing the expression of genes involved in synthesis and uptake, leading to a decrease in intracellular cholesterol content. These results suggested that this class of compounds could potentially be used to modulate brain cholesterol metabolism in pathological conditions. We used primary cultures of mice neurons and astrocytes treated with the pan-HDACi trichostatin A, and with class-specific HDAC inhibitors, namely, the class I inhibitor Mocetinostat (MGCD0103) and the class II (IIa) inhibitor MC1568, to evaluate the mRNA levels of a panel of genes involved in cholesterol synthesis, up-take, transport and elimination. Although we could only partially recapitulate the results obtained in the neuroblastoma cells, we could identify class I HDACs as major modulators of neuronal cholesterol homeostasis. In addition, when comparing the expression profiles of neurons and astrocytes, we identified Mvk, Cyp46a1 and Apoe as genes that are affected by both TSA and MGCD0103 in both cells. In order to further dissect the role of class I HDACs we specifically silenced class I HDACs by transfecting TALEN specific plasmids in SH-SY5Y neuroblastoma cells. Our results point to a role of class I HDACs as therapeutic targets both in neuroblastoma and neurodegenerative disorders with disturbances in cholesterol homeostasis. Acknowledgements This work was supported by FEDER and national funds from Fundação para a Ciência e Tecnologia (FCT) (UID/DTP/04138/2013 and fellowship SFRH/BPD/95855/2013 to MJN). Keywords: cholesterol metabolism, HDACs, HDAC inhibitors, neurodegeneration, Neuroblastoma Conference: XVI Meeting of the Portuguese Society for Neuroscience (SPN2019), Lisboa, Portugal, 30 May - 1 Jun, 2019. Presentation Type: Poster presentation Topic: Cellular and Molecular Neurosciences Citation: Nunes M, Costa D, Mendes De Almeida R, Castro-Caldas M, Gama M and Rodrigues E (2019). Targeting cholesterol metabolism in neuroblastoma by acting on HDACs. Front. Cell. Neurosci. Conference Abstract: XVI Meeting of the Portuguese Society for Neuroscience (SPN2019). doi: 10.3389/conf.fncel.2019.01.00012 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 16 Apr 2019; Published Online: 27 Sep 2019. * Correspondence: Prof. Elsa Rodrigues, Research Institute for Medicines (iMed.ULisboa), Lisboa, Portugal, elsa.rodrigues@ff.ulisboa.pt Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Maria João Nunes Daniela Costa Rita Mendes De Almeida Margarida Castro-Caldas Maria João Gama Elsa Rodrigues Google Maria João Nunes Daniela Costa Rita Mendes De Almeida Margarida Castro-Caldas Maria João Gama Elsa Rodrigues Google Scholar Maria João Nunes Daniela Costa Rita Mendes De Almeida Margarida Castro-Caldas Maria João Gama Elsa Rodrigues PubMed Maria João Nunes Daniela Costa Rita Mendes De Almeida Margarida Castro-Caldas Maria João Gama Elsa Rodrigues Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Novel therapeutic approaches to high-risk neuroblastoma targeting cancer metabolism

Novel therapeutic approaches to high-risk neuroblastoma targeting cancer metabolism

LncRNA RMRP silence curbs neonatal neuroblastoma progression by regulating microRNA-206/tachykinin-1 receptor axis via inactivating extracellular signal-regulated kinases

ABSTRACTBackground: Neuroblastoma is the commonest malignancy in neonates. Long non-coding RNA (lncRNA) RNA component of mitochondrial RNA processing endoribonuclease (RMRP) has been reported to be an oncogenic factor in some malignancies. However, its roles and molecular mechanisms in neuroblastoma progression are poor defined.Methods: The expression of RMRP, microRNA-206 (miR-206), and tachykinin-1 receptor (TACR1) mRNA was measured by RT-qPCR assay. Protein levels of TACR1, phosphorylated extracellular signal-regulated kinases (ERK) 1/2 (p-ERK1/2) and ERK1/2 were detected by western blot assay. Cell proliferation was assessed by CCK-8 and colony formation assays. Cell migratory and invasive capacities were determined using Transwell migration and invasion assays. The interaction between miR-206 and RMRP or TACR1 was verified by luciferase assay. The roles and molecular mechanisms of RMRP knockdown on the growth of neuroblastoma xenografts were examined in vivo.Results: RMRP was highly expressed in neuroblastoma tissues. RMRP knockdown inhibited proliferation, migration and invasion in neuroblastoma cells. Moreover, TACR1 was a target of miR-206 and RMRP performed as a molecular sponge of miR-206 to sequester miR-206 from TACR1 in neuroblastoma cells. TACR1 overexpression abrogated the inhibitory effect of RMRP downregulation on neuroblastoma cell progression by activating ERK1/2 pathway. Inhibition of TACR1 and ERK1/2 pathway abated RMRP-mediated pro-proliferation effect in neuroblastoma cells. RMRP knockdown hindered neuroblastoma xenograft growth by regulating miR-206/TACR1 axis via inactivating ERK1/2 pathway in vivo.Conclusion: RMRP knockdown hindered the tumorigenesis and progression of neuroblastoma by regulating miR-206/TACR1 axis via inactivating ERK1/2 pathway, hinting a potential therapeutic target for neuroblastoma.

Kinetically selective and potent inhibitors of HDAC8.

Histone deacetylase 8 (HDAC8) is an established and validated target for T-cell lymphoma and childhood neuroblastoma. The active site binding pocket of HDAC8 is highly conserved among all zinc-containing representatives of the histone deacetylase (HDAC) family. This explains that most HDACs are unselectively recognized by similar inhibitors featuring a zinc binding group (ZBG), a hydrophobic linker and a head group. In the light of this difficulty, the creation of isoenzyme-selectivity is one of the major challenges in the development of HDAC inhibitors. In a series of trifluoromethylketone inhibitors of HDAC8 compound 10 shows a distinct binding mechanism and a dramatically increased residence time (RT) providing kinetic selectivity against HDAC4. Combining the binding kinetics results with computational docking and binding site flexibility analysis suggests that 10 occupies the conserved catalytic site as well as an adjacent transient sub-pocket of HDAC8.

EPAS1/HIF2α correlates with features of low-risk neuroblastoma and with adrenal chromaffin cell differentiation during sympathoadrenal development

The hypoxia inducible transcription factor EPAS1/HIF2α has been described as an oncogene and a potential therapeutic target in neuroblastoma. Our analysis of several neuroblastoma tumour expression datasets does not support an oncogenic role, instead EPAS1 expression is associated with better patient outcome and characteristics of low-risk tumours. Treatment with HIF2α inhibitors did not block in vitro neuroblastoma cell proliferation nor xenograft growth. In addition, we analysed single cell sequencing data sets from the developing mouse sympathoadrenal lineage, wherein expression of Epas1 was a strong predictor of differentiated adrenal chromaffin cells and negatively correlated with progenitor characteristics. This was reflected in neuroblastoma tumours wherein genes co-expressed with Epas1 during sympathoadrenal development strongly predicts favourable patient outcome and features of low-risk tumours. Thus, our analysis suggest that with the current available data EPAS1/HIF2α should not be classified as a neuroblastoma oncogene and is less likely to represent a suitable drug target in this disease.

Influence of glucose transporter 1 activity inhibition on neuroblastoma in vitro

Most cancer cells predominantly produce their energy through a high rate of glycolysis in the presence of abundant oxygen. Glycolysis has become a target of anticancer strategies. Previous researches showed that glucose transporter 1 (GLUT1) inhibitor is effective as anticancer agents. This study assessed the effects of the selective GLUT1 inhibitor WZB117 on regulation of neuroblastoma (NB) cell line SH-SY5Y viability, cell cycle and glycolysis in vitro. SH-SY5Y cells were grown and treated with WZB117 for up to 72 h and then subjected to cell viability, qRT-PCR, Western blot and flow cytometry analysis. Level of ATP and LDH was also analyzed. The result showed that WZB117 treatment reduced tumor cells viability, downregulated level of GLUT1 protein. Moreover, WZB117 treatment arrested tumor cells at the G0–G1 phase of the cell cycle, induced tumor cells to undergo necrosis instead of apoptosis. In addition, WZB117 treatment downregulated the levels of intracellular ATP, LDH and glycolytic enzymes. Thus, WZB117-induced GLUT1 inhibition suppressed tumor cell growth, induced cell cycle arrest and reduced glycolysis metabolites in NB cells in vitro. This study suggested that GLUT1 can be used as a potential therapeutic target for NB.

LDHA in Neuroblastoma Is Associated with Poor Outcome and Its Depletion Decreases Neuroblastoma Growth Independent of Aerobic Glycolysis.

Purpose: To investigate whether lactate dehydrogenase A (LDHA), an important component of the LDH tetramer crucial for aerobic glycolysis, is associated with patient outcome and constitutes a therapeutic target in neuroblastoma (NB).Experimental Design: Expression of LDHA mRNA and protein was determined in 709 and 110 NB patient samples, respectively, and correlated with survival and risk factors. LDHA and LDHB were depleted in human NB cell lines by CRISPR/Cas9 and shRNA, respectively, and aerobic glycolysis, clonogenicity, and tumorigenicity were determined. Expression of LDHA in relation to MYCN was measured in NB cell lines and in the TH-MYCN NB mouse model.Results: Expression of LDHA, both on the mRNA and the protein level, was significantly and independently associated with decreased patient survival. Predominant cytoplasmic localization of LDHA protein was associated with poor outcome. Amplification and expression of MYCN did not correlate with expression of LDHA in NB cell lines or TH-MYCN mice, respectively. Knockout of LDHA inhibited clonogenicity, tumorigenicity, and tumor growth without abolishing LDH activity or significantly decreasing aerobic glycolysis. Concomitant depletion of LDHA and the isoform LDHB ablated clonogenicity while not abrogating LDH activity or decreasing aerobic glycolysis. The isoform LDHC was not expressed.Conclusions: High expression of LDHA is independently associated with outcome of NB, and NB cells can be inhibited by depletion of LDHA or LDHB. This inhibition appears to be unrelated to LDH activity and aerobic glycolysis. Thus, investigations of inhibitory mechanisms beyond attenuation of aerobic glycolysis are warranted, both in NB and normal cells. Clin Cancer Res; 24(22); 5772-83. ©2018 AACR.

Tumor-suppressive function of SIRT4 in neuroblastoma through mitochondrial damage.

BackgroundSIRT4 is a member of the sirtuin family of nicotinamide adenine dinucleotide-dependent enzymes located in the mitochondria, and is involved in regulating energy metabolism, stress response, and cellular lifespan in mammalian cells. However, its function in human neuroblastoma (NB) remains unexplored.MethodsExpression of SIRT4 in 158 pairs of human NB tumor tissues and adjacent normal tissues collected from March 2009 to October 2012 was analyzed by immunohistochemistry, Western blotting, and real-time fluorescence quantitative PCR. For in vitro study, SIRT4 was overexpressed in SH-SY5Y, SK-N-BE, and IMR-32 cells to study the effects of SIRT4 expression on proliferation, invasion, and migration of human NB cells and on mitochondrial function.ResultsSIRT4 gene expression in human NB tumor tissues was significantly lower than that in adjacent normal tissues (P<0.001). SIRT4 expression was lower in NB patients with higher International Neuroblastoma Staging System stage (P=0.018), with lymph node metastasis, than patients without lymph node metastasis (P<0.001). Survival times of NB patients with low expression of SIRT4 were significantly shorter than those of patients with high expression of SIRT4 (P=0.0036). Overexpression of SIRT4 significantly reduced the proliferation, invasion, and migration ability of NB cells as well as mitochondrial energy production, and caused SIRT1 upregulation and mitochondrial damage in NB cells.ConclusionSIRT4 exhibits a tumor suppressor function in human NB and inhibits mitochondrial metabolism and SIRT1 expression in tumor cells, thereby reducing the energy metabolism of tumor cells. These results suggest that SIRT4 may be a new therapeutic target for human NB.

GZD2202, a novel TrkB inhibitor, suppresses BDNF-mediated proliferation and metastasis in neuroblastoma models

Collective data suggest tropomyosin-related kinase B (TrkB), which is correlated with the growth, migration and poor prognosis of neuroblastoma (NB), is a potential target for NB target therapy. Several Phase I/II pan-Trk inhibitors display impressive clinical outcomes but still no drug has been approved for general use. In this paper, we report a novel structural TrkB inhibitor GZD2202, a structural derivative of our previously identified DDR1 antagonists. GZD2202 demonstrates a moderate selectivity between Trk B/C and TrkA. GZD2202 suppresses the brain-derived neurotrophic factor (BDNF) -mediated TrkB signalling pathway, proliferation, migration and invasion in SH-SY5Y-TrkB neuroblastoma cells, and causes about 36.1% growth inhibition in a SH-SY5Y-TrkB neuroblastoma xenograft model.