The Potential Functional Roles of NME1 Histidine Kinase Activity in Neuroblastoma Pathogenesis

Neuroblastoma is the most common extracranial solid tumor in childhood. Gain of chromosome 17q material is found in >60% of neuroblastoma tumors and is associated with poor patient prognosis. The NME1 gene is located in the 17q21.3 region, and high NME1 expression is correlated with poor neuroblastoma patient outcomes. However, the functional roles and signaling activity of NME1 in neuroblastoma cells and tumors are unknown. NME1 and NME2 have been shown to possess histidine (His) kinase activity. Using anti-1- and 3-pHis specific monoclonal antibodies and polyclonal anti-pH118 NME1/2 antibodies, we demonstrated the presence of pH118-NME1/2 and multiple additional pHis-containing proteins in all tested neuroblastoma cell lines and in xenograft neuroblastoma tumors, supporting the presence of histidine kinase activity in neuroblastoma cells and demonstrating the potential significance of histidine kinase signaling in neuroblastoma pathogenesis. We have also demonstrated associations between NME1 expression and neuroblastoma cell migration and differentiation. Our demonstration of NME1 histidine phosphorylation in neuroblastoma and of the potential role of NME1 in neuroblastoma cell migration and differentiation suggest a functional role for NME1 in neuroblastoma pathogenesis and open the possibility of identifying new therapeutic targets and developing novel approaches to neuroblastoma therapy.

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Plasticity in Neuroblastoma Cell Identity Defines a Noradrenergic-to-Mesenchymal Transition (NMT)

Cancers ◽

10.3390/cancers13122904 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2904

Author(s):

Margot Gautier ◽

Cécile Thirant ◽

Olivier Delattre ◽

Isabelle Janoueix-Lerosey

Keyword(s):

Epithelial To Mesenchymal Transition ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

Biochemical Properties ◽

Poor Overall Survival ◽

Cell Identity ◽

Mesenchymal Transition ◽

Neuroblastoma Cell Lines

Neuroblastoma, a pediatric cancer of the peripheral sympathetic nervous system, is characterized by an important clinical heterogeneity, and high-risk tumors are associated with a poor overall survival. Neuroblastoma cells may present with diverse morphological and biochemical properties in vitro, and seminal observations suggested that interconversion between two phenotypes called N-type and S-type may occur. In 2017, two main studies provided novel insights into these subtypes through the characterization of the transcriptomic and epigenetic landscapes of a panel of neuroblastoma cell lines. In this review, we focus on the available data that define neuroblastoma cell identity and propose to use the term noradrenergic (NOR) and mesenchymal (MES) to refer to these identities. We also address the question of transdifferentiation between both states and suggest that the plasticity between the NOR identity and the MES identity defines a noradrenergic-to-mesenchymal transition, reminiscent of but different from the well-established epithelial-to-mesenchymal transition.

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Regulation of c-myb expression in human neuroblastoma cells during retinoic acid-induced differentiation.

Molecular and Cellular Biology ◽

10.1128/mcb.8.4.1677 ◽

1988 ◽

Vol 8 (4) ◽

pp. 1677-1683 ◽

Cited By ~ 54

Author(s):

C J Thiele ◽

P S Cohen ◽

M A Israel

Keyword(s):

Retinoic Acid ◽

Fetal Development ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

Human Neuroblastoma Cell ◽

Human Neuroblastoma Cells ◽

Human Neuroblastoma ◽

Hematopoietic Lineage ◽

Specific Manner ◽

Neuroblastoma Cell Lines

We detected expression of the c-myb proto-oncogene, which was initially thought to be expressed in a tissue-specific manner in cells of hematopoietic lineage, in human tissues of neuronal origin. Since the level of c-myb expression declined during fetal development, we studied the regulation of its expression in human neuroblastoma cell lines induced to differentiate by retinoic acid. The expression of c-myb declined during the maturation of neuroblastoma cells, and this change was mediated by a decrease in c-myb transcription.

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Itchy E3 ubiquitin-protein ligase promotes neuroblastoma progression in vitro and in vivo

Medical Research ◽

10.6913/mr.0304.03 ◽

2021 ◽

Vol 3 (4) ◽

pp. 12-24

Author(s):

Mabao YUAN ◽

Hanjiao HANG ◽

Lubin YAN ◽

Xuanjie HUANG ◽

Ziyang SANG ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Lines ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

Luciferase Reporter ◽

Ubiquitin Protein Ligase ◽

Neuroblastoma Cell Lines ◽

Dual Luciferase Reporter Assay

[Objective] Neuroblastoma is the most common pediatric neuroendocrine tumor. Patients with high-risk neuroblastoma have poor clinical outcomes. Understanding the mechanisms underlying neuroblastoma progression could help identify potential therapeutic targets. This study aimed to explore the roles of itchy E3 ubiquitin-protein ligase (ITCH) in neuroblastoma progression using neuroblastoma cell lines and xenograft models of neuroblastoma. [Methods] ITCH-silencing or overexpressing neuroblastoma cells were established using two different human neuroblastoma cell lines, SK-N-AS and SH-SY5Y. In vitro and in vivo experiments were carried out to determine the effects of ITCH on neuroblastoma cell behaviors. The dual-luciferase reporter assay and co-transfection experiments were applied to determine the interaction of ITCH and miR-145-5p during neuroblastoma progression. [Results] In both cell lines, ITCH overexpression significantly promotes the proliferation, migration, and invasion capacities of neuroblastoma cells, while ITCH silencing with ShITCH suppressed neuroblastoma cell proliferation and induced apoptosis. Moreover, overexpression of ITCH decreased 51% and 54% the protein expressions of large tumor suppressor kinase 1 (LATS1), and inhibited 59% and 66% the phosphorylation of Yes-associated protein (YAP), concomitant with 2.02-fold and 2.56-fold increased expressions of cell proliferation marker Ki67 and 2.51-fold and 2.26-fold elevated levels of anti-apoptosis marker Bcl2 in SK-N-AS and SH-SY5Y cells, respectively. The dual-luciferase reporter assay demonstrated that ITCH interacted with miR-145-5p. Further in vitro and xenograft experiments showed that ITCH negatively affected the tumor-suppressive effect of miR-145-5p. [Conclusion] ITCH promotes neuroblastoma cell proliferation and metastasis by inhibiting LATS1 and promoting YAP nuclear translocation.

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Activation of cell migration via morphological changes in focal adhesions depends on shear stress in MYCN-amplified neuroblastoma cells

Journal of The Royal Society Interface ◽

10.1098/rsif.2018.0934 ◽

2019 ◽

Vol 16 (152) ◽

pp. 20180934

Author(s):

Takumi Hiraiwa ◽

Takahiro G. Yamada ◽

Norihisa Miki ◽

Akira Funahashi ◽

Noriko Hiroi

Keyword(s):

Shear Stress ◽

Cell Migration ◽

Cell Motility ◽

Morphological Changes ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

Focal Adhesions ◽

Human Neuroblastoma Cell ◽

Human Neuroblastoma ◽

The Relationship

Neuroblastoma is the most common solid tumour of childhood, and it metastasizes to distant organs. However, the mechanism of metastasis, which generally depends on the cell motility of the neuroblastoma, remains unclear. In many solid tumours, it has been reported that shear stress promotes metastasis. Here, we investigated the relationship between shear stress and cell motility in the MYCN-amplified human neuroblastoma cell line IMR32, using a microfluidic device. We confirmed that most of the cells migrated downstream, and cell motility increased dramatically when the cells were exposed to a shear stress of 0.4 Pa, equivalent to that expected in vivo . We observed that the morphological features of focal adhesion were changed under a shear stress of 0.4 Pa. We also investigated the relationship between malignancy and the motility of IMR32 cells under shear stress. Decreasing the expression of MYCN in IMR32 cells via siRNA transfection inhibited cell motility by a shear stress of 0.4 Pa. These results suggest that MYCN-amplified neuroblastoma cells under high shear stress migrate to distant organs due to high cell motility, allowing cell migration to lymphatic vessels and venules.

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GX15–070 (Obatoclax), a Bcl-2 family proteins inhibitor engenders apoptosis and pro-survival autophagy and increases Chemosensitivity in neuroblastoma

BMC Cancer ◽

10.1186/s12885-019-6195-y ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 6

Author(s):

Sonia Cournoyer ◽

Anissa Addioui ◽

Assila Belounis ◽

Mona Beaunoyer ◽

Carine Nyalendo ◽

...

Keyword(s):

Cell Lines ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

Distant Metastases ◽

Caspase Inhibitor ◽

Pediatric Tumor ◽

Apoptotic Protein ◽

Synergistic Cytotoxicity ◽

Neuroblastoma Cell Lines

Abstract Background Neuroblastoma (NB) is a frequent pediatric tumor associated with poor prognosis. The disregulation of Bcl-2, an anti-apoptotic protein, is crucial for the tumoral development and chemoresistance. Autophagy is also implicated in tumor cell survival and chemoresistance. The aim of our study was to demonstrate therapeutic efficiency of GX 15–070, a pan-Bcl-2 family inhibitor, used alone and in combination with conventional drugs or with hydroxychloroquine (HCQ), an autophagy inhibitor. Methods Five neuroblastoma cell lines were tested for the cytotoxic activity of GX 15–070 alone or in combination with cisplatin, doxorubicin, HCQ or Z-VAD-FMK a broad-spectrum caspase inhibitor. Apoptosis and autophagy levels were studied by western-blot and FACS. Orthotopic injections were performed on NOD/LtSz-scid/IL-2Rgamma null mice that were treated with either GX 15–070 alone or in combination with HCQ. Results Synergistic cytotoxicity was observed for the drug combination in all of the 5 neuroblastoma cell lines tested, including MYCN amplified lines and in cancer stem cells. GX 15–070 significantly increased apoptosis and autophagy in neuroblastoma cells as evidenced by increased levels of the autophagy marker, LC3-II. Inhibition of autophagy by HCQ, further increased the cytotoxicity of this combinatorial treatment, suggesting that autophagy induced by these agent plays a cytoprotective role. In vivo, GX 15–070 combined with HCQ significantly decreased the growth of the tumor and the number of distant metastases. Conclusions Based on the synergistic effect of HCQ and GX 15–070 observed in this study, the combination of these two drugs may be utilized as a new therapeutic approach for neuroblastoma.

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ALT neuroblastoma chemoresistance due to telomere dysfunction–induced ATM activation is reversible with ATM inhibitor AZD0156

Science Translational Medicine ◽

10.1126/scitranslmed.abd5750 ◽

2021 ◽

Vol 13 (607) ◽

pp. eabd5750

Author(s):

Balakrishna Koneru ◽

Ahsan Farooqi ◽

Thinh H. Nguyen ◽

Wan Hsi Chen ◽

Ashly Hindle ◽

...

Keyword(s):

Cell Lines ◽

Ataxia Telangiectasia ◽

De Novo ◽

Clinical Stage ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

Telomere Dysfunction ◽

Neuroblastoma Cell Lines

Cancers overcome replicative immortality by activating either telomerase or an alternative lengthening of telomeres (ALT) mechanism. ALT occurs in ~25% of high-risk neuroblastomas, and progression in patients with ALT neuroblastoma during or after front-line therapy is frequent and often fatal. Temozolomide + irinotecan is commonly used as salvage therapy for neuroblastoma. Patient-derived cell lines and xenografts established from patients with relapsed ALT neuroblastoma demonstrated de novo resistance to temozolomide + irinotecan [SN-38 in vitro, P < 0.05; in vivo mouse event-free survival (EFS), P < 0.0001] vs. telomerase-positive neuroblastomas. We observed that ALT neuroblastoma cells manifested constitutive ataxia-telangiectasia mutated (ATM) activation due to spontaneous telomere dysfunction which was not observed in telomerase-positive neuroblastoma cells. We demonstrated that induction of telomere dysfunction resulted in ATM activation that, in turn, conferred resistance to temozolomide + SN-38 (4.2-fold change in IC50, P < 0.001). ATM knockdown (shRNA) or inhibition using a clinical-stage small-molecule inhibitor (AZD0156) reversed resistance to temozolomide + irinotecan in ALT neuroblastoma cell lines in vitro (P < 0.001) and in four ALT xenografts in vivo (EFS, P < 0.0001). AZD0156 showed modest to no enhancement of temozolomide + irinotecan activity in telomerase-positive neuroblastoma cell lines and xenografts. Ataxia telangiectasia and Rad3 related (ATR) inhibition using AZD6738 did not enhance temozolomide + SN-38 activity in ALT neuroblastoma cells. Thus, ALT neuroblastoma chemotherapy resistance occurs via ATM activation and is reversible with ATM inhibitor AZD0156. Combining AZD0156 with temozolomide + irinotecan warrants clinical testing for neuroblastoma.

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Amplified N-myc in human neuroblastoma cells is often arranged as clustered tandem repeats of differently recombined DNA.

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.4903 ◽

1989 ◽

Vol 9 (11) ◽

pp. 4903-4913 ◽

Cited By ~ 72

Author(s):

L C Amler ◽

M Schwab

Keyword(s):

Tandem Repeats ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

Selective Advantage ◽

Central Position ◽

Coding Region ◽

Human Neuroblastoma Cells ◽

Human Neuroblastoma ◽

Low Degrees ◽

Neuroblastoma Cell Lines

Human neuroblastoma cells often carry amplified DNA encompassing the gene N-myc. Amplified N-myc has been found localized in "double minutes" in direct tumor cell preparations. In contrast, later passages carried amplified N-myc almost exclusively within a single homogeneously staining chromosomal region located at a chromosomal site different from the normal location of N-myc. We used pulsed field gel electrophoresis to define the structural arrangement of the amplified DNA. Long-range mapping was facilitated by the presence of several sites for rare cutting restriction endonucleases in the 5' region of N-myc. Amplified DNAs of different neuroblastoma cell lines were heterogeneous in size and had undergone recombination at various distances from N-myc. N-myc occupied a central position within the amplified DNA, and in no case was the coding region affected by recombination. Among neuroblastoma cells, varying proportions of amplified DNA (in some instances close to 100%) consisted of multiple tandem arrays of DNA segments ranging in size from 100 to 700 kilobase pairs. Tumor cells with low degrees of amplification revealed regions of amplified DNA in excess of 1,500 kilobase pairs without apparent rearrangement. Our observations, in concert with the cytogenetic findings, suggest a model of gene amplification which involves unscheduled DNA replication, recombination, and formation of extrachromosomal DNA followed by integration into a chromosome and subsequent in situ multiplication. The central position which N-myc occupies within the amplified sequences and the lack of recombination within the coding region of N-mc indicate that N-myc rather than other genetic information provides the selective advantage for retention of the amplified DNA.

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Different Subcellular Localization of ALCAM Molecules in Neuroblastoma: Association with Relapse

Analytical Cellular Pathology ◽

10.1155/2010/160417 ◽

2010 ◽

Vol 32 (1-2) ◽

pp. 77-86

Author(s):

Maria Valeria Corrias ◽

Claudio Gambini ◽

Andrea Gregorio ◽

Michela Croce ◽

Gaia Barisione ◽

...

Keyword(s):

Nervous System ◽

Subcellular Localization ◽

Cell Lines ◽

System Development ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

Nervous System Development ◽

Primary Tumors ◽

Neuroblastoma Cell Lines

Background: The Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD), involved in nervous system development, has been linked to tumor progression and metastasis in several tumors. No information is available on ALCAM expression in neuroblastoma, a childhood neoplasia originating from the sympathetic nervous system.Methods: ALCAM expression was analysed by immunofluorescence and immunohistochemistry on differentiated neuroblastoma cell lines and on archival specimens of stroma-poor, not MYCN amplified, resectable neuroblastoma tumors, respectively.Results: ALCAM is variously expressed in neuroblastoma cell lines, is shed by metalloproteases and is cleaved by ADAM17/TACE in vitro. ALCAM is expressed in neuroblastoma primary tumors with diverse patterns of subcellular localization and is highly expressed in the neuropil area in a subgroup of cases. Tumor specimens showing high expression of ALCAM at the membrane of the neuroblast body or low levels in the neuropil area are associated with relapse (P = 0.044 and P < 0.0001, respectively). In vitro differentiated neuroblastoma cells show strong ALCAM expression on neurites, suggesting that ALCAM expression in the neuropil is related to a differentiated phenotype.Conclusions: Assessment of ALCAM localization by immunohistochemistry may help to identify patients who, in the absence of negative prognostic factors, are at risk of relapse and require a more careful follow-up.

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

Cancers ◽

10.3390/cancers11020202 ◽

2019 ◽

Vol 11 (2) ◽

pp. 202 ◽

Cited By ~ 1

Author(s):

Conny Tümmler ◽

Gianina Dumitriu ◽

Malin Wickström ◽

Peter Coopman ◽

Andrey Valkov ◽

...

Keyword(s):

Cell Lines ◽

Inflammatory Diseases ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

Chemotherapeutic Drugs ◽

Akt Phosphorylation ◽

Risk Patients ◽

Syk Inhibitor ◽

Neuroblastoma Cell Lines

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 613606 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.

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NME/NM23/NDPK and Histidine Phosphorylation

International Journal of Molecular Sciences ◽

10.3390/ijms21165848 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5848

Author(s):

Kevin Adam ◽

Jia Ning ◽

Jeffrey Reina ◽

Tony Hunter

Keyword(s):

Histidine Kinase ◽

Kinase Activity ◽

Current Knowledge ◽

Family Members ◽

Nucleoside Diphosphate Kinase ◽

Molecular Tools ◽

New Methods ◽

Functional Aspects ◽

Histidine Phosphorylation ◽

Nucleoside Diphosphate Kinases

The NME (Non-metastatic) family members, also known as NDPKs (nucleoside diphosphate kinases), were originally identified and studied for their nucleoside diphosphate kinase activities. This family of kinases is extremely well conserved through evolution, being found in prokaryotes and eukaryotes, but also diverges enough to create a range of complexity, with homologous members having distinct functions in cells. In addition to nucleoside diphosphate kinase activity, some family members are reported to possess protein-histidine kinase activity, which, because of the lability of phosphohistidine, has been difficult to study due to the experimental challenges and lack of molecular tools. However, over the past few years, new methods to investigate this unstable modification and histidine kinase activity have been reported and scientific interest in this area is growing rapidly. This review presents a global overview of our current knowledge of the NME family and histidine phosphorylation, highlighting the underappreciated protein-histidine kinase activity of NME family members, specifically in human cells. In parallel, information about the structural and functional aspects of the NME family, and the knowns and unknowns of histidine kinase involvement in cell signaling are summarized.

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