Undifferentiated Neuroblastoma Research Articles

Ageritin from poplar mushrooms: scale-up purification and cytotoxicity towards undifferentiated and differentiated SH-SY5Y cells

Ageritin is the first reported ribotoxin-like protein from basidiomycetes fungi. It can induce ribosomal integrity damage and translation block, and interferes with mitochondrial redox activity of some glioma and neuroblastoma cell lines. Herein, Ageritin has been investigated as a valuable neurotoxin towards either undifferentiated or retinoic acid (RA)-differentiated SH-SY5Y neuroblastoma cells showing a selective cell toxicity against undifferentiated cells. MTT and sulforhodamine B (SRB) assays highlighted that Ageritin markedly decreases the mitochondrial redox activity and viability of undifferentiated cells, meanwhile inducing evident morphological changes eliciting neuronal-like appearance in these cells. Data from lactate dehydrogenase release assay, cytofluorimetric analysis and caspase-3 enzymatic activity measurement suggest that Ageritin promotes cell death through a caspase-dependent apoptotic pathway. The Z-VAD-FMK caspase inhibitor was able to prevent this apoptotic pathway activation. Based on the interesting behaviour of Ageritin vs. SH-SY5Y cells, the development of a scale-up procedure to obtain the purified protein in larger amounts (yield 2.5 mg per 100 g) has been optimized.

Reduced Extracellular Matrix Stiffness Prompts SH-SY5Y Cell Softening and Actin Turnover To Selectively Increase Aβ(1-42) Endocytosis.

Alzheimer's disease (AD), the most common neurodegenerative disorder, is characterized by the extracellular deposition of dense amyloid beta plaques. Emerging evidence suggests that the production of these plaques is initiated by the intracellular uptake and lysosomal preconcentration of the amyloid-beta (Aβ) peptide. All previous endocytosis studies assess Aβ uptake with cells plated on traditional tissue culture plastic; however, brain tissue is distinctly soft with a low-kPa stiffness. Use of an ultrastiff plastic/glass substrate prompts a mechanosensitive response (increased cell spreading, cell stiffness, and membrane tension) that potentially distorts a cell's endocytic behavior from that observed in vivo or in a more physiologically relevant mechanical environment. Our studies demonstrate substrate stiffness significantly modifies the behavior of undifferentiated SH-SY5Y neuroblastoma, where cells plated on soft (∼1 kPa) substrates display a rounded morphology, decreased actin polymerization, reduced adhesion (decreased β1 integrin expression), and reduced cell stiffness compared to cells plated on tissue culture plastic. Moreover, these neuroblastoma on softer substrates display a preferential increase in the uptake of the Aβ(1-42) compared to Aβ(1-40), while both isoforms display a clear stiffness-dependent increase of uptake relative to cells plated on plastic. Considering the brain is a soft tissue that continues to soften with age, this mechanosensitive endocytosis of Aβ has significant implications for understanding age-related neurodegeneration and the mechanism behind Aβ uptake and fibril production. Overall, identifying these physical factors that contribute to the pathology of AD may offer novel avenues of therapeutic intervention.

Differentiation-Dependent Effects of a New Recombinant Manganese Superoxide Dismutase on Human SK-N-BE Neuron-Like Cells

We have recently isolated a new isoform of recombinant manganese superoxide dismutase (rMnSOD) which provides a potent antitumor activity and strongly counteracts the occurrence of oxidative stress and tissue inflammation. This isoform, in addition to the enzymatic action common to all SODs, also shows special functional and structural properties, essentially due to the presence of a first leader peptide that allows the protein to enter easily into cells. Among endogenous antioxidants, SOD constitutes the first line of natural defence against pathological effects induced by an excess of free radicals. Here, we firstly describe the effects of our rMnSOD administration on the proliferant and malignant undifferentiated human neuroblastoma SK-N-BE cell line. Moreover, we also test the effects of rMnSOD in the all trans retinoic-differentiated SK-N-BE neuron-like cells, a quiescent "not malignant" model. While rMnSOD showed an antitumor activity on proliferating cells, a poor sensitivity to rMnSOD overload in retinoid-differentiated neuron-like cells was observed. However, in the latter case, in presence of experimental-induced oxidative stress, overcharge of rMnSOD enhanced the oxidant effects, through an increase of H2O2 due to low activity of both catalase and glutathione peroxidase. In conclusion, our data show that rMnSOD treatment exerts differential effects, which depend upon both cell differentiation and redox balance, addressing attention to the potential use of the recombinant enzyme on differentiated neurons. These facts ultimately pave the way for further preclinical studies aimed at evaluation of rMnSOD effects in models of neurodegenerative diseases.

Indibulin dampens microtubule dynamics and produces synergistic antiproliferative effect with vinblastine in MCF-7 cells: Implications in cancer chemotherapy

Indibulin, a synthetic inhibitor of tubulin assembly, has shown promising anticancer activity with a minimal neurotoxicity in preclinical animal studies and in Phase I clinical trials for cancer chemotherapy. Using time-lapse confocal microscopy, we show that indibulin dampens the dynamic instability of individual microtubules in live breast cancer cells. Indibulin treatment also perturbed the localization of end-binding proteins at the growing microtubule ends in MCF-7 cells. Indibulin reduced inter-kinetochoric tension, produced aberrant spindles, activated mitotic checkpoint proteins Mad2 and BubR1, and induced mitotic arrest in MCF-7 cells. Indibulin-treated MCF-7 cells underwent apoptosis-mediated cell death. Further, the combination of indibulin with an anticancer drug vinblastine was found to exert synergistic cytotoxic effects on MCF-7 cells. Interestingly, indibulin displayed a stronger effect on the undifferentiated neuroblastoma (SH-SY5Y) cells than the differentiated neuronal cells. Unlike indibulin, vinblastine and colchicine produced similar depolymerizing effects on microtubules in both differentiated and undifferentiated SH-SY5Y cells. The data indicated a possibility that indibulin may reduce chemotherapy-induced peripheral neuropathy in cancer patients.

MicroRNA-494 Favors HO-1 Expression in Neuroblastoma Cells Exposed to Oxidative Stress in a Bach1-Independent Way.

Heme oxygenase 1 (HO-1) is crucially involved in cell adaptation to oxidative stress and has been demonstrated to play an important role in cancer progression and resistance to therapies. We recently highlighted that undifferentiated neuroblastoma (NB) cells are prone to counteract oxidative stress through the induction of HO-1. Conversely, differentiated NB cells were more sensitive to oxidative stress since HO-1 was scarcely upregulated. In this work, we investigated the role played by miR-494, which has been proved to be involved in cancer biology and in the modulation of oxidative stress, in the upregulation of HO-1. We showed that NB differentiation downregulates miR-494 level. In addition, endogenous miR-494 inhibition in undifferentiated cells impairs HO-1 induction in response to exposure to 500 µM H2O2, reducing the number of viable cells. The analysis of Bach1 expression did not reveal any significant modifications in any experimental conditions tested, proving that the impairment of HO-1 induction observed in cells treated with miR-494 inhibitor and exposed to H2O2 is independent from Bach1. Our results underline the role played by miR-494 in favoring HO-1 induction and cell adaptation to oxidative stress and contribute to the discovery of new potential pharmacological targets to improve anticancer therapies.

Enhanced Intratumoral Delivery of SN38 as a Tocopherol Oxyacetate Prodrug Using Nanoparticles in a Neuroblastoma Xenograft Model.

Purpose: Currently, <50% of high-risk pediatric solid tumors like neuroblastoma can be cured, and many survivors experience serious or life-threatening toxicities, so more effective, less toxic therapy is needed. One approach is to target drugs to tumors using nanoparticles, which take advantage of the enhanced permeability of tumor vasculature.Experimental Design: SN38, the active metabolite of irinotecan (CPT-11), is a potent therapeutic agent that is readily encapsulated in polymeric nanoparticles. Tocopherol oxyacetate (TOA) is a hydrophobic mitocan that was linked to SN38 to significantly increase hydrophobicity and enhance nanoparticle retention. We treated neuroblastomas with SN38-TOA nanoparticles and compared the efficacy with the parent prodrug CPT-11 using a mouse xenograft model.Results: Nanoparticle treatment induced prolonged event-free survival (EFS) in most mice, compared with CPT-11. This was shown for both SH-SY5Y and IMR-32 neuroblastoma xenografts. Enhanced efficacy was likely due to increased and sustained drug levels of SN38 in the tumor compared with conventional CPT-11 delivery. Interestingly, when recurrent CPT-11-treated tumors were re-treated with SN38-TOA nanoparticles, the tumors transformed from undifferentiated neuroblastomas to maturing ganglioneuroblastomas. Furthermore, these tumors were infiltrated with Schwann cells of mouse origin, which may have contributed to the differentiated histology.Conclusions: Nanoparticle delivery of SN38-TOA produced increased drug delivery and prolonged EFS compared to conventional delivery of CPT-11. Also, lower total dose and drug entrapment in nanoparticles during circulation should decrease toxicity. We propose that nanoparticle-based delivery of a rationally designed prodrug is an attractive approach to enhance chemotherapeutic efficacy in pediatric and adult tumors. Clin Cancer Res; 24(11); 2585-93. ©2018 AACR.

The absence of a novel intron 19-retaining ALK transcript (ALK-I19) and MYCN amplification correlates with an excellent clinical outcome in neuroblastoma patients.

ALK missense mutations are detected in 8% of neuroblastoma (NB) tumors at diagnosis and confer gain-of-function oncogenic effects. The mechanisms by which the expression of wild-type or mutant ALK, which is detectable in the majority of cases, is regulated are not well understood. We have identified a novel ALK transcript characterized by the retention of intron 19 (ALK-I19). ALK-I19 was detected in 4/4 NB cell lines, but not other non-NB cells with ALK aberrations. The functional significance of ALK-I19 was determined by specific siRNA knockdown of this transcript, which resulted in substantially decreased expression of the fully-spliced ALK transcripts (FS-ALK) and a significant reduction in cell growth. We also demonstrate that ALK-I19 is a precursor of FS-ALK. ALK-I19 was detected in 14/37 (38%) tumors from patients with newly diagnosed NB. ALK-I19 expression correlated with undifferentiated histology and strong ALK protein expression detectable by immunohistochemistry. Importantly, patients with tumors that did not express ALK-I19 and lacked MYCN amplification had an excellent clinical outcome, with 19/19 patients survived at 5-years. In conclusion, ALK-I19 is a novel ALK transcript that likely represents a marker of undifferentiated NB cells. The absence of ALK-I19 and MYCN amplification is a useful prognostic marker for NB patients.

Expression of DHA-Metabolizing Enzyme Alox15 is Regulated by Selective Histone Acetylation in Neuroblastoma Cells

The omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA) is enriched in neural membranes of the CNS, and recent studies have shown a role of DHA metabolism by 15-lipoxygenase-1 (Alox15) in prefrontal cortex resolvin D1 formation, hippocampo-prefrontal cortical long-term-potentiation, spatial working memory, and anti-nociception/anxiety. In this study, we elucidated epigenetic regulation of Alox15 via histone modifications in neuron-like cells. Treatment of undifferentiated SH-SY5Y human neuroblastoma cells with the histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate significantly increased Alox15 mRNA expression. Moreover, Alox15 expression was markedly upregulated by Class I HDAC inhibitors, MS-275 and depsipeptide. Co-treatment of undifferentiated SH-SY5Y cells with the p300 histone acetyltransferase (HAT) inhibitor C646 and TSA or sodium butyrate showed that p300 HAT inhibition modulated TSA or sodium butyrate-induced Alox15 upregulation. Differentiation of SH-SY5Y cells with retinoic acid resulted in increased neurite outgrowth and Alox15 mRNA expression, while co-treatment with the p300 HAT inhibitor C646 and retinoic acid modulated the increases, indicating a role of p300 HAT in differentiation-associated Alox15 upregulation. Increasing Alox15 expression was found in primary murine cortical neurons during development from 3 to 10 days-in-vitro, reaching high levels of expression by 10 days-in-vitro—when Alox15 was not further upregulated by HDAC inhibition. Together, results indicate regulation of Alox15 mRNA expression in neuroblastoma cells by histone modifications, and increasing Alox15 expression in differentiating neurons. It is possible that one of the environmental influences on the immature brain that can affect cognition and memory, may take the form of epigenetic effects on Alox15 and metabolites of DHA.

Legumain correlates with neuroblastoma differentiation and can be used in prodrug design.

Neuroblastoma (NB) is a highly malignant solid tumor in children. The cysteine endopeptidase legumain is expressed in adult solid tumors, but its expression in NB has not been examined. In this study, we assayed legumain expression in two NB cell lines and in microarrays of tumor tissues collected from 46 children with undifferentiated NB, differentiated NB, and ganglioneuroblastoma. Correlation analyses showed that legumain was expressed in all NB cell lines tested and that expression correlated with the degree of tumor differentiation. The efficacy, specificity, and toxicity of EMC-AANL-DOX, a novel doxorubicin-based legumain-activated prodrug, were then evaluated in mouse model of NB. Compared with DOX, EMC-AANL-DOX showed greater inhibition of tumor growth and a lower toxicity at high doses. Neither leukocyte or platelet counts nor renal function or cardiac anatomy differed significantly between the EMC-AANL-DOX and control groups (p>.05), suggesting that the prodrug caused minimal bone marrow depression and did not induce renal or cardiac damage. The good specificity and efficacy of EMC-AANL-DOX and low toxicity recommend its use in the treatment of NB. Correlation analyses of NB differentiation and legumain expression may reveal a novel anti-tumor-related functions of the drug and a new strategy for the treatment of pediatric solid tumors.

The involvement of autophagy and cytoskeletal regulation in TDCIPP-induced SH-SY5Y cell differentiation

Exposure and toxicity to organophosphate-based flame retardants are an increasing health concern. Neurons appear to be particularly vulnerable to the effects of these chemicals. For example, in vitro studies have shown that tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) induces apoptosis and autophagy in neural cells. In the present study, we investigated the cell biological mechanisms of TDCIPP-induced neurotoxicity using undifferentiated human SH-SY5Y neuroblastoma cells as a model. Interestingly, TDCIPP treatment promoted differentiation in SH-SY5Y cells, which displayed various alterations including neurite elongation, an expansion of the numbers of neurite-bearing cells, and an increase in expression of cytoskeletal components normally enriched in neurons. Furthermore, the upregulation of microtubule-associated protein light chain 3, the degradation of p62/sequestosome 1, and the formation of autophagosomes occurred in treated cells, suggesting that TDCIPP exposure induces autophagy. However, pretreatment with the autophagy inhibitor 3-methyladenine suppressed TDCIPP-induced autophagy and reduced expression of the aforementioned cytoskeletal components. This correlated with a reduction in neurite outgrowth and numbers of neurite-bearing cells. Taken together, these results indicate that autophagy might promote TDCIPP-induced SH-SY5Y cell differentiation, which leads to an increase in expression of cytoskeletal components and neurite outgrowth. This study offers key insights into the mechanisms of neurotoxicity associated with this commonly used organophosphate.

Immunohistochemical Profile of MYC Protein in Pediatric Small Round Blue Cell Tumors.

Deregulation of MYC oncoprotein in cancers can result from multiple oncogenic mechanisms. Although MYC translocations define Burkitt lymphoma and MYC protein expression is a poor prognostic factor in undifferentiated neuroblastomas, the distribution of MYC protein (c-MYC) across other pediatric small round blue cell tumors (SRBCT) has not been well characterized. We undertook this study to assess MYC protein expression in a large cohort of pediatric lymphomas, sarcomas, and other SRBCT. Tissue microarrays containing 302 SRBCT were successfully evaluated by immunohistochemistry using anti-MYC clone Y69, with nuclear positivity scored as 0%, 1%-25%, 26%-50%, 51%-75%, or 76%-100%. MYC protein staining of >50% of lesional cells was identified in 60% of Burkitt lymphomas, 50% of B lymphoblastic lymphomas, 33% of T lymphoblastic lymphomas, 31% of rhabdomyosarcomas, 33% of Ewing sarcomas, and 25% of soft tissue sarcomas, not otherwise specified. Only 14% of neuroblastomas showed >50% staining, and of these, if known, MYCN was not amplified. No cases of Wilms tumor, synovial sarcoma, or desmoplastic small round cell tumor had >50% staining. Recurrences and metastases often had the same percentage of MYC staining (15/30). In conclusion, MYC protein exhibited variable expression across and within pediatric SRBCT subtypes. Overall, these findings provide a baseline for MYC expression in pediatric SRBCT and suggest that there may be multiple mechanisms of MYC upregulation in these different neoplasms.

Results of neuroblastoma treatment in Lithuania: a single centre experience.

Background.Neuroblastoma (NB) is the most common extracranial solid tumour in children. This is a very rare disease with heterogeneous biology varying from complete spontaneous regression to a highly aggressive tumour responsible for 15% of malignancy-related death in early childhood. Analyses of survival rates in Europe have shown a considerable difference between Northern/Western and Eastern European countries. Treatment results of NB in Lithuania have never been analyzed.Aim.To assess the survival rate of children with NB according to initial spread of the disease, age at diagnosis, the MYCN amplification, risk group, and treatment period.Patients and methods.A retrospective single-centre analysis of patients’ records was performed. Children diagnosed and treated for NB between 2000 and 2015 at the Centre of Paediatric Oncology and Haematology of the Children’s Hospital, Affiliate of Vilnius University Hospital Santaros Klinikos were included. The patients were divided into three groups according to the spread of the disease: group 1 – patients with local NB older than 12 years of age; group 2 – stage IV patients, also called the M stage; group 3 – infants with stages 4S and MS. The patients were stratified into three risk groups – low, intermediate and high risk. Estimates of five-year overall survival (OS5y) were calculated using the Kaplan-Meier method comparing survival probability according to spread of the disease, age at diagnosis, the MYCN amplification, risk group and treatment period (2000–2007 vs 2008–2015).Results.Overall 60 children (31 girls and 29 boys) with NB were included. The median age at diagnosis was 1.87 years (ranged from 4 days to 15 years). Seventy-eight percent of cases were found to be differentiated or undifferentiated NB, 22% – ganglioneuroblastoma. The local form of the disease was predominant: 57% (34/60) of patients were allocated to the group 1, 37% (22/60) with initial metastatic disease were assigned to group 2, and infants with 4S or MS stage comprising 7% (4/60) allocated to group 3, respectively. The probability of OS5y for the entire cohort was 71% with the median follow-up of 8.8 ± 4.8 years. The probability of OS5y for local disease (group 1) was significantly higher compared to metastatic disease (group 2) (94% vs. 34%, p = 0.001, respectively) as well as for infants compared to children older than 12 months at the time of diagnosis (90% vs 60%, p = 0.009, respectively). The MYCN gene amplification had a negative influence on OS5y, with 78% of MYCN-negative patients surviving in comparison to 40% of MYCN-positive patients who did not survive (p = 0.153). The high-risk patients had significantly worse OS5y than children with intermediated or low risk (35% vs. 82% vs. 100%, respectively, p = 0.001). Comparison of OS5y between two treatment periods in the entire patient population revealed a non-significant increase in survival from 66% in the 2000–2007 period to 82% in the 2008–2015 period (p = 0.291), mostly due to a dramatic improvement achieved for high-risk patients whose survival rate increased from 9% in the 2000–2007 period to 70% in the 2008–2015 period (p = 0.009).Conclusions.There was a slight predominance of low-risk patients, probably due to a higher number of infants. A better probability of OS5y was confirmed in infants with local disease and in MYCN-negative patients. The OS5y for children treated for NB at our institution over 16 years increased from 66% in the 2000–2007 period to 82% in the 2008–2015 period with the most significant improvement achieved for high risk patients. The current survival rate of children treated for NB at our institution is in line with the reported numbers in Northern and Western European countries.

231 - Neuroblastoma Cell Response to Oxidative Stress Is Impaired by Retinoic Acid-Induced Differentiation: Role of HO-1

The modulation of redox balance is able to drive different cell functions, from differentiation to proliferation. In a previous work, we showed that all-trans retinoic acid (ATRA) induced differentiation of SH-SY5Y neuroblastoma (NB) cells through the generation of reactive oxygen species (ROS) [Nitti et al Cell Signal 2010]. However, ATRA-differentiated NB cells resulted more sensitive to the further unbalance of redox state in comparison with undifferentiated ones, as shown in response to glycoxydative stress [Nitti et al Neurosci Letters 2007]. Heme oxygenase 1 (HO-1), induced by the transcription factor Nrf2, plays a crucial role in cell adaptation to oxidative stress through the generation of bilirubin, ferritin and carbon monoxide with antioxidant and anti-inflammatory properties. In this work, focusing on Nrf2/HO-1 axis, we investigated the ability of undifferentiated and ATRA-differentiated NB cells to balance oxidative stress. SH-SY5Y exposure to 100-500 μM H2O2 decreased cell viability significantly more in ATRA- differentiated cells than in the undifferentiated ones. The expression of GCLM and GCLC, key enzymes in the synthesis of GSH, was not modified by H2O2 treatment in any condition. However, after H2O2 treatment heme oxygenase 1 (HO-1) expression and Nrf2 binding to HO-1 promoter were significantly reduced in differentiated cells in comparison to the undifferentiated ones. In addition, exposure to bilirubin prevented cell death induced by H2O2 in differentiated cells. Our data show that retinoic acid-dependent differentiation impairs Nrf2-dependent HO-1 induction in response to H2O2 and, by reducing the availability of endogenous bilirubin, decreases cell adaption to oxidative stress. Nrf2 nuclear translocation and post-transcriptional modifications are under investigation. Grant from MIUR-PRIN20125S38FA and Genoa University.

Infectivity of Immature Neurons to Zika Virus: A Link to Congenital Zika Syndrome

BackgroundEpidemiological data strongly suggest that microcephaly cases in Brazil are associated with the ongoing epidemic of Zika virus (ZIKV). In order to further solidify the possible link, we investigated the infectivity of ZIKV using various neuroblastoma (NB) cell lines. MethodsSix undifferentiated, two terminally differentiated and two retinoic acid (RA) –induced, partially differentiated cell lines were exposed to ZIKV strain PRVABC59, which is genetically similar to the French Polynesia strain, with 97–100% genetic homology to the current ZIKV strain found in Brazil. All infections were confirmed by real-time PCR (RT-qPCR), immunofluorescence assay (IFA) probing with anti-flavivirus E antibody, and evaluation of cytopathic effects. FindingsZIKV infected all six undifferentiated NB cell lines. In five out of six NB cell lines, between 90 and 70% cells were positive by IFA whereas for one cell line, CCL-127, ~80% of cells were positive for ZIKV as determined by IFA but showed persistent infection. Two differentiated cell lines, JFEN and T-268, were highly resistant to ZIKV with <1% of the cells being susceptible, as determined by IFA and confirmed by qRT-PCR. Two retinoic acid (RA)-induced NB partially differentiated cell lines showed no difference in permissiveness as compared to their undifferentiated mother cell lines. InterpretationThese findings strengthen the reported association between high incidences of microcephaly and ZIKV infection in newborns in Brazil. Our results suggest that the undifferentiated neurons are highly permissive to ZIKV infection, as one would expect during the early stages of neurogenesis in fetal brains; whereas differentiated neurons, representative of adult brain neurons, are relatively resistant to the virus, which explains the rare occurrence of neurological complications in adults infected with ZIKV. Our studies confirm the neurotropism of the ZIKV strain closely related to the current epidemic in Latin America.

Comparative study of the neurotoxicological effects of tramadol and tapentadol in SH-SY5Y cells.

Opioid therapy and abuse are increasing, justifying the need to study their toxicity and underlying mechanisms. Given opioid pharmacodynamics at the central nervous system, the analysis of toxic effects in neuronal models gains particular relevance. The aim of this study was to compare the toxicological effects of acute exposure to tramadol and tapentadol in the undifferentiated human SH-SY5Y neuroblastoma cell line. Upon exposure to tramadol and tapentadol concentrations up to 600μM, cell toxicity was assessed through evaluation of oxidative stress, mitochondrial and metabolic alterations, as well as cell viability and death mechanisms through necrosis or apoptosis, and related signalling. Tapentadol was observed to trigger much more prominent toxic effects than tramadol, ultimately leading to energy deficit and cell death. Cell death was shown to predominantly occur through necrosis, with no alterations in membrane potential or in cytochrome c release. Both drugs were shown to stimulate glucose uptake and to cause ATP depletion, due to changes in the expression of energy metabolism enzymes. The toxicity mechanisms in such a neuronal model are relevant to understand adverse reactions to these opioids and to contribute to dose adjustment in order to avoid neurological damage.

Distinct effect of CacyBP/SIP on the ERK1/2-CREB-BDNF pathway in undifferentiated and differentiated neuroblastoma NB2a cells

CacyBP/SIP, a protein expressed to high extent in the brain, has been shown to act as ERK1/2 phosphatase in vitro and in cultured cells. It has been demonstrated recently that CacyBP/SIP can modulate the activity of some transcription factors in neurons and glioma cells. In the present work we have examined the effect of CacyBP/SIP overexpression and silencing on the phosphorylation/activity of ERK1/2 (pERK1/2) and CREB (pCREB) and on the level of BDNF mRNA in differentiated and undifferentiated neuroblastoma NB2a cells. We have shown that in undifferentiated cells the amount of pERK1/2 decreased upon CacyBP/SIP overexpression. Further studies have shown that the activity of CREB and the level of BDNF mRNA, downstream effectors of the ERK1/2 signaling pathway, also depended on the CacyBP/SIP level and strictly matched the level of pERK1/2. Interestingly, in differentiated NB2a cells, overexpression of CacyBP/SIP appeared to have a distinct effect on the pERK1/2 level from that observed in undifferentiated cells. Subsequent studies have revealed that distinct function of CacyBP/SIP in undifferentiated and differentiated NB2a cells might be due to changes in its posttranslational modifications and protein ligands. Altogether, our studies suggest that CacyBP/SIP is involved in the ERK1/2-CREB-BDNF pathway and that it might regulate this pathway depending on the stage of NB2a cell differentiation.

Identification of DDX39A as a Potential Biomarker for Unfavorable Neuroblastoma Using a Proteomic Approach.

Malignant potential in unfavorable neuroblastoma (NB) is dependent on an undifferentiated status. The aim of this study was to identify a novel biomarker associated with the undifferentiated status of NB in vitro and to evaluate its prognostic implication. Shotgun proteomic analysis was performed in undifferentiated and all trans-retinoic acid induced differentiated NB cells in vitro. An identified protein was verified by multiple reaction monitoring (MRM) and evaluated by Western blot analysis. Immunohistochemistry (IHC) was used to examine the expression of the identified protein in 33 primary NB tissues. Twelve proteins, including ATP-dependent RNA helicase (DDX39A), were only detected in undifferentiated NB cells. A peptide of DDX39A was detected at a significantly higher level in undifferentiated IMR-32 (P = 0.002) and LA-N-1 (P < 0.001) cells by MRM. Western blot analysis revealed that DDX39A expression was significantly higher in undifferentiated IMR-32 (P = 0.02) and LA-N-1 (P = 0.025) cells. IHC demonstrated that DDX39A was highly expressed in the primary tumor tissues of patients with poor prognosis, and univariate and multivariate survival analyses showed that DDX39A expression could be an independent unfavorable prognostic factor (P = 0.027). DDX39A is a potential biomarker for unfavorable NB using a proteomic approach. Evaluation of DDX39A protein expression in NB tumor tissues may provide complementary prognostic information for further subclassification of these tumors.

Abstract A02: The epigenetic modifier CHAF1A opposes neuroblastoma differentiation via metabolic reprogramming

Abstract Background. Neuroblastoma (NB) arises from embryonal neural crest secondary to a block in differentiation and long-term survival inversely correlates with degree of neuronal differentiation. Metabolic rewiring is an important feature of undifferentiated NB tumors. Interestingly, MYCN down regulation precedes differentiation of NB cells induced by retinoic acid (RA) and genetic MYCN silencing leads to neuronal differentiation. We have recently demonstrated a novel function for the histone chaperone CHAF1A in blocking NB differentiation. CHAF1A is a subunit of the Chromatin Assembly Factor-1 (CAF1), which regulates H3K9-trimethylation and DNA methylation. Methods. Morphological and molecular evaluations of NB differentiation were assessed upon CHAF1A silencing in RA-sensitive and RA-resistant lines by neurite outgrowth assays and Q-PCR for mature neuronal markers. Gene expression profiling of CHAF1A knockdown and control cell lines was performed on the Affymetrix U133+2.0 arrays. GSEA (Gene Set Enrichment Analysis) defined the transcriptional response to CHAF1A silencing in RA sensitive lines. Quantitative-PCR assays were used to validate the most significant enriched metabolic gene sets and MYCN western blotting was performed at different time points after CHAF1A silencing. Results. CHAFIA suppression in MYCN-amplified RA-sensitive and RA-resistant lines results in NB differentiation and inhibition of cell proliferation. CHAF1A silencing significantly (nominal p-value &amp;lt;0.05 and FDR q-value &amp;lt;0.25) enriches for cell metabolism pathways (valine, leucine, and isoleucine degradation, glutamate metabolism and insulin pathways) and suppresses pathways with known oncogenic function in neuroblastoma (KRAS, ALK, Akt and BMI1). Q-PCR studies confirmed the regulation by CHAF1A of gene sets regulating glucose and glutamine metabolism. Importantly, MYCN protein levels were profoundly suppressed upon CHAF1A silencing. Conclusions. We have shown that differentiation of NB cells upon CHAF1A silencing: 1) induces metabolic reprogramming by decreasing glucose and glutamine uptakes, 2) inhibits cell proliferation, and 3) reduces MYCN protein levels. We hypothesize that CHAF1A plays a critical role in the MYCN-mediated metabolic reprogramming of undifferentiated NB tumors. Future studies will further dissect this link and guide the development of novel differentiating therapies for high-risk NB. Citation Format: Eveline Barbieri, Zaowen Chen, Mirthala Moreno-Smith, Jason M. Shohet. The epigenetic modifier CHAF1A opposes neuroblastoma differentiation via metabolic reprogramming. [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 A02.

Abstract 1628: Neuropeptide Y (NPY) and its receptor expression in neuroblastoma patients - associations with disease prognosis and patients’ survival

Abstract Neuropeptide Y (NPY) is a sympathetic neurotransmitter, abundantly expressed in neuroblastoma (NB). Previously, we have shown that NPY, acting via its Y2 receptor (Y2R), stimulates proliferation of NB cells and tumor vascularization, while inducible Y5 receptor (Y5R) promotes tumor cell survival and chemoresistance. The aim of the current study was to assess the prognostic value of NPY and its receptor expression in NB. We have tested corresponding samples of RNA, tissue sections and serum from 87 NB patients at various stages of the disease, obtained from the Children's Oncology Group. Samples were analyzed in terms of NPY system expression (NPY, Y2R and Y5R). Protein levels and mRNA in tumor tissue were quantified by immunohistochemistry (IHC) and real time-PCR, respectively, while NPY concentration in serum was measured by ELISA. The expression of other factors implicated in NB development and progression (MYCN, ALK, TrkA III, TrkB and BDNF) were detected on mRNA levels by real time-PCR. For each of the categorical prognostic variables, a Wilcoxon rank-sum or Fisher's exact test were administered to compare NPY and receptor values between groups, and a log-rank test was performed to compare the event-free survival and overall survival between groups. 1) NPY mRNA was detectable in 100% of analyzed tumors. IHC staining revealed that NPY accumulated intracellularly in differentiating and maturing cells, while in undifferentiated NBs a significant amount of the peptide was observed in extracellular spaces, suggesting its free release. In line with this observation, serum concentrations of NPY were higher in the subset of NB patients with undifferentiated and poorly differentiated tumors, as compared to those with differentiating NBs (p-value = 0.03). High serum concentrations of NPY strongly correlated with several adverse prognostic factors (Stage 4, high risk, diploidy, and unfavorable histology) (p-value&amp;lt;0.03) and worse survival (p-value&amp;lt;0.04). 2) Y2R was expressed mainly in undifferentiated NB cells. mRNA of this receptor was detectable in 100% of cases and its levels positively correlated with MYCN expression 3) mRNA of Y5R was detectable in 84% of tumors assessed and correlated with expression of BDNF and its TrkB receptor, factors associated with poor prognosis. High Y5R protein levels marked a population of invasive NB cells. This study validated NPY and its receptors as targets for NB therapy. Serum NPY was the most highly prognostic variable, found to be associated with five NB prognostic factors. Higher NPY levels were correlated with a more detrimental disease phenotype. Citation Format: Susana Galli, Jason Tilan, Arlene Naranjo, Collin Van Ryn, Chao Yang, Jessica Tsuei, Emily Trinh, Joanna B. Kitlinska. Neuropeptide Y (NPY) and its receptor expression in neuroblastoma patients - associations with disease prognosis and patients’ survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1628. doi:10.1158/1538-7445.AM2015-1628

Abstract 3955: A primary cell-based assay to identify initiating oncogenic events in neuroblastoma

Abstract The pediatric cancer neuroblastoma (NBL) forms from immature cells of the sympathetic nervous system (SNS). A prominent category of high-risk NBL is MYCN amplification with loss of heterozygosity (LOH) at 1p36. This LOH includes the chromatin remodeling factor, CHD5, a proposed tumor suppressor of NBL and can include ARID1A, a component of the SWI/SNF chromatin remodeling complex. To identify genetic changes that cooperate with MYCN amplification in initiating NBL we developed an assay based on oncogenic transformation of neural crest cells (NCCs), the progenitors of the SNS. Isolation of NCCs from E9.5 mouse embryos resulted in a population that was ∼90% positive for the NCC markers p75, Sox10, and Ascl1, suggesting a highly enriched population. Next, we tested the ability of N-Myc infected NCCs to form tumors in vivo. In pilot experiments using NCCs with compromised p53, we obtained subcutaneous tumors following injection of NCCs in nude mice. Resulting tumors were diffusely positive for neuronal markers such as synaptophysin, MAP2, and Tyrosine hydroxylase (TH). Tumors also contained focal areas of divergent differentiation characteristic of melanoma, Schwannoma, and osteosarcoma, all cancers that can arise from NCC lineages. To our knowledge, this is the first report of cancer from transformed primary neural crest cells. To more closely model NBL genetics, we replaced loss of p53 with shRNA to knockdown CHD5 and ARID1A prior to N-Myc infection. Injection of the resulting NCCs also resulted in tumor formation in nude mice. Tumors were uniformly positive for neuronal markers including TH and Ascl1, as well as Phox2B, a marker of undifferentiated NBL, and with no evidence of other cancers. Future studies in understanding the interactions of N-Myc overexpression with loss of these chromatin remodeling factors should further increase our understanding of NBL development. Citation Format: Rachelle Olsen, Joel Otero, Zhirong Yin, Jesus Garcia-Lopez, Kevin Freeman. A primary cell-based assay to identify initiating oncogenic events in neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3955. doi:10.1158/1538-7445.AM2015-3955