Expression In Neuroblastoma Cells Research Articles

2,3,7,8-Tetrachlorodibenzo-p-dioxin and kynurenine induce Parkin expression in neuroblastoma cells through different signaling pathways mediated by the aryl hydrocarbon receptor

Parkin regulates protein degradation and mitophagy in dopaminergic neurons. Deficiencies in Parkin expression or function lead to cellular stress, cell degeneration, and the death of dopaminergic neurons, which promotes Parkinson's disease. In contrast, Parkin overexpression promotes neuronal survival. Therefore, the mechanisms of Parkin upregulation are crucial to understand. We describe here the molecular mechanism of AHR-mediated Parkin regulation in human SH-SY5Y neuroblastoma cells. Specifically, we report that the human Parkin gene (PRKN) is transcriptionally upregulated by the aryl hydrocarbon receptor (AHR) through two different selective ligand-dependent pathways. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a stress-inducing AHR ligand, indirectly promotes PRKN transcription by inducing ATF4 expression via TCDD-mediated endoplasmic reticulum (ER) stress. In contrast, kynurenine, a nontoxic AHR agonist, induces PRKN transcription by promoting AHR binding to the PRKN promoter without activating ER stress.Our results demonstrate that AHR activation may be a potential pharmacological pathway to induce human Parkin, but such a strategy must carefully consider the choice of AHR ligand to avoid neurotoxic side effects.

LncRNA ZNF674-AS1 drives cell growth and inhibits cisplatin-induced pyroptosis via up-regulating CA9 in neuroblastoma

Neuroblastoma (NB) is a challenging pediatric extracranial solid tumor characterized by a poor prognosis and resistance to chemotherapy. Identifying targets to enhance chemotherapy sensitivity in NB is of utmost importance. Increasing evidence implicates long noncoding RNAs (lncRNAs) play important roles in cancer, but their functional roles remain largely unexplored. Here, we analyzed our RNA sequencing data and identified the upregulated lncRNA ZNF674-AS1 in chemotherapy non-responsive NB patients. Elevated ZNF674-AS1 expression is associated with poor prognosis and high-risk NB. Importantly, targeting ZNF674-AS1 expression in NB cells suppressed tumor growth in vivo. Further functional studies have revealed that ZNF674-AS1 constrains cisplatin sensitivity by suppressing pyroptosis and promoting cell proliferation. Moreover, ZNF674-AS1 primarily relies on CA9 to fulfill its functions on cisplatin resistance. High CA9 levels were associated with high-risk NB and predicted poor patient outcomes. Mechanistically, ZNF674-AS1 directly interacted with the RNA binding protein IGF2BP3 to enhance the stability of CA9 mRNA by binding with CA9 transcript, leading to elevated CA9 expression. As a novel regulator of CA9, IGF2BP3 positively upregulated CA9 expression. Together, these results expand our understanding of the cancer-associated function of lncRNAs, highlighting the ZNF674-AS1/IGF2BP3/CA9 axis as a constituting regulatory mode in NB tumor growth and cisplatin resistance. These insights reveal the pivotal role of ZNF674-AS1 inhibition in recovering cisplatin sensitivity, thus providing potential therapeutic targets for NB treatment.

Abstract LB_C20: A tumor-associated chemokine attracts CCR4-expressing CD4+ cells to foster immune repression and tumor aggressiveness in MYCN-driven neuroblastoma

Abstract Neuroblastoma, especially those with aberrant MYCN activation, often harbor an immunosuppressive microenvironment to fuel malignant growth and trigger treatment resistance. Despite this knowledge, there are no effective strategies to tackle this problem. Here we combined analyses of human neuroblastoma with live tracking and functional studies of the tumor microenvironment (TME) in zebrafish. We identified a tumor-associated chemokine (TAC) as a key driver of MYCN-mediated immunosuppression and neuroblastoma aggression. Taking advantage of in vivo zebrafish models of MYCN-driven neuroblastoma that resemble human high-risk disease, we demonstrated that TAC overexpression in zebrafish neural crest cells induces an immunosuppressive TME while promoting rapid tumor onset and progression. We also demonstrated that as early as the premalignant stage, tumor cells secrete TAC to attract CCR4-expressing Cd4+ cells to induce immunosuppression and tumor aggression. In addition, genetically depleting Cd4+ T regulatory cells abolishes the immunorestrictive and pro-tumorigenic effects of TAC. We showed that human MYCN-activated neuroblastoma upregulates and secretes TAC, which is a reliable predictor of poor patient survival. Analysis of primary patient samples demonstrates a strong positive association between MYCN and TAC expression in neuroblastoma cells together with the enrichment of FOXP3+ T cells in the TME. Our work provides the first example that MYCN can activate TAC to allure CD4+ immune cells to the TME and incite immunosuppression, positioning TAC as a potential novel immunotherapeutic target for the treatment of MYCN-driven high-risk neuroblastoma. Citation Format: Xiaodan Qin, Andrew Lam, Xu Zhang, Satyaki Sengupta, J. Bryan Iorgulescu, Sanjukta Das, Zhenwei Zhou, Tao Zuo, Grace K. Meara, Madison Rager, Alexander E. Floru, Hongru Ni, Chinyere Kemet, Divya Veerapaneni, Daniel Kashy, Liang Lin, Kenneth Lloyd, Lauren Kwok, Kaylee S. Smith, Raghavendar T. Nagaraju, Rob Meijers, Craig Ceol, Ching-Ti Liu, Sanda Alexandrescu, Catherine J. Wu, Derin B. Keskin, Rani E. George, Hui Feng. A tumor-associated chemokine attracts CCR4-expressing CD4+ cells to foster immune repression and tumor aggressiveness in MYCN-driven neuroblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C20.

A miR-137–Related Biological Pathway of Risk for Schizophrenia Is Associated With Human Brain Emotion Processing

BackgroundmiR-137 is a microRNA involved in brain development, regulating neurogenesis and neuronal maturation. Genome-wide association studies have implicated miR-137 in schizophrenia risk but do not explain its involvement in brain function and underlying biology. Polygenic risk for schizophrenia mediated by miR-137 targets is associated with working memory, although other evidence points to emotion processing. We characterized the functional brain correlates of miR-137 target genes associated with schizophrenia while disentangling previously reported associations of miR-137 targets with working memory and emotion processing. MethodsUsing RNA sequencing data from postmortem prefrontal cortex (N = 522), we identified a coexpression gene set enriched for miR-137 targets and schizophrenia risk genes. We validated the relationship of this set to miR-137 in vitro by manipulating miR-137 expression in neuroblastoma cells. We translated this gene set into polygenic scores of coexpression prediction and associated them with functional magnetic resonance imaging activation in healthy volunteers (n1 = 214; n2 = 136; n3 = 2075; n4 = 1800) and with short-term treatment response in patients with schizophrenia (N = 427). ResultsIn 4652 human participants, we found that 1) schizophrenia risk genes were coexpressed in a biologically validated set enriched for miR-137 targets; 2) increased expression of miR-137 target risk genes was mediated by low prefrontal miR-137 expression; 3) alleles that predict greater gene set coexpression were associated with greater prefrontal activation during emotion processing in 3 independent healthy cohorts (n1, n2, n3) in interaction with age (n4); and 4) these alleles predicted less improvement in negative symptoms following antipsychotic treatment in patients with schizophrenia. ConclusionsThe functional translation of miR-137 target gene expression linked with schizophrenia involves the neural substrates of emotion processing.

RNA N6-methyladenosine reader IGF2BP3 interacts with MYCN and facilitates neuroblastoma cell proliferation

Neuroblastoma (NB) is a kind of typical life-threatening extracranial tumor in children. N6-methyladenosine (m6A) modification is closely related to multiple cancer pathological processes. Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) is a top-ranked prognostic risk gene in NB; however, its function is uncertain. The expression of m6A-associated enzymes in patients with NB was analyzed using the Gene Expression Omnibus (GEO) and Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database. The IGF2BP3 level in NB cell lines and primary samples was tested using quantitative real-time polymerase chain reaction (qRT-PCR), western blot method, and immunohistochemical analysis. The IGF2BP3 function in cell proliferation was clarified based on many functional in vitro and in vivo experiments. The interaction between IGF2BP3 and N-myc was researched via RNA immunoprecipitation (RIP), m6A RNA immunoprecipitation (MeRIP), and chromatin immunoprecipitation (ChIP) assays. The 16 m6A-regulated enzymes in NB were researched, and the result indicated that IGF2BP3 overexpression was related to cancer progression, COG risk, and survival based on the GEO and TARGET databases. Besides, the IGF2BP3 and MYCN levels were positively correlated. IGF2BP3 expression levels increased in MYCN-amplified NB clinical samples and cells. Knockdown of IGF2BP3 inhibited N-myc expression and NB cell proliferation in vitro and in vivo. IGF2BP3 regulates MYCN RNA stability by modifying m6A. In addition, we demonstrated that N-myc is a transcription factor that directly promotes IGF2BP3 expression in NB cells. IGF2BP3 regulates the proliferation of NB cells via m6A modification of MYCN. N-myc also acts as a transcription factor that regulates IGF2BP3 expression. A positive feedback loop between IGF2BP3 and N-myc facilitates NB cell proliferation.

Calreticulin Expression Controls Cellular Redox, Stemness, and Radiosensitivity to Function as a Novel Adjuvant for Radiotherapy in Neuroblastoma.

Radiotherapy (RT) is currently only used in children with high-risk neuroblastoma (NB) due to concerns of long-term side effects as well as lack of effective adjuvant. Calreticulin (CALR) has served distinct physiological roles in cancer malignancies; nonetheless, impact of radiation on chaperones and molecular roles they play remains largely unknown. In present study, we systemically analyzed correlation between CALR and NB cells of different malignancies to investigate potential role of CALR in mediating radioresistance of NB. Our data revealed that more malignant NB cells are correlated to lower CALR expression, greater radioresistance, and elevated stemness as indicated by colony- and neurospheroid-forming abilities and vice versa. Of note, manipulating CALR expression in NB cells of varying endogenous CALR expression manifested changes in not only stemness but also radioresistant properties of those NB cells. Further, CALR overexpression resulted in greatly enhanced ROS and led to increased secretion of proinflammatory cytokines. Importantly, growth of NB tumors was significantly hampered by CALR overexpression and was synergistically ablated when RT was also administered. Collectively, our current study unraveled a new notion of utilizing CALR expression in malignant NB to diminish cancer stemness and mitigate radioresistance to achieve favorable therapeutic outcome for NB.

Safety and efficacy of dinutuximab in the treatment of neuroblastoma: A review.

Dinutuximab, which is a monoclonal antibody targeting GD2 expressed in neuroblasts, improves survival when included in the therapy regimen. This article reviews the importance of dinutuximab in managing neuroblastoma (NB). Dinutuximab targets high levels of GD2 expression in NB cells, thus increasing event-free survival when used in the maintenance therapy of high-risk patients with NB. Although several collaborative studies have set the standard of care for maintenance therapy, the long-term follow-up and continuous evaluation of the use of antibodies and the co-administration of other pharmacological or immunomodulatory drugs remain to be studied. Trials have shown that the use of dinutuximab for maintenance therapy can prolong the time before the first relapse and improve overall survival. However, there is uncertainty in the function of cytokines co-administered with dinutuximab, which may lead to increased toxicity without additional benefits. Recent studies on relapsed and refractory NB have shown the potential efficacy of dinutuximab. Further research is required to properly incorporate Dinutuximab in current treatment modalities.

Anti-GRP-R monoclonal antibody antitumor therapy against neuroblastoma.

Standard treatment for patients with high-risk neuroblastoma remains multimodal therapy including chemoradiation, surgical resection, and autologous stem cell rescue. Immunotherapy has demonstrated success in treating many types of cancers; however, its use in pediatric solid tumors has been limited by low tumor mutation burdens. Gastrin-releasing peptide receptor (GRP-R) is overexpressed in numerous malignancies, including poorly-differentiated neuroblastoma. Monoclonal antibodies (mAbs) to GRP-R have yet to be developed but could serve as a potential novel immunotherapy. This preclinical study aims to evaluate the efficacy of a novel GRP-R mAb immunotherapy against neuroblastoma. We established four candidate anti-GRP-R mAbs by screening a single-chain variable fragment (scFv) library. GRP-R mAb-1 demonstrated the highest efficacy with the lowest EC50 at 4.607 ng/ml against GRP-R expressing neuroblastoma cells, blocked the GRP-ligand activation of GRP-R and its downstream PI3K/AKT signaling. This resulted in functional inhibition of cell proliferation and anchorage-independent growth, indicating that mAb-1 has an antagonist inhibitory role on GRP-R. To examine the antibody-dependent cellular cytotoxicity (ADCC) of GRP-R mAb-1 on neuroblastoma, we co-cultured neuroblastoma cells with natural killer (NK) cells versus GRP-R mAb-1 treatment alone. GRP-R mAb-1 mediated ADCC effects on neuroblastoma cells and induced release of IFNγ by NK cells under co-culture conditions in vitro. The cytotoxic effects of mAb-1 were confirmed with the secretion of cytotoxic granzyme B from NK cells and the reduction of mitotic tumor cells in vivo using a murine tumor xenograft model. In summary, GRP-R mAb-1 demonstrated efficacious anti-tumor effects on neuroblastoma cells in preclinical models. Importantly, GRP-R mAb-1 may be an efficacious, novel immunotherapy in the treatment of high-risk neuroblastoma patients.

BCI, an inhibitor of the DUSP1 and DUSP6 dual specificity phosphatases, enhances P2X7 receptor expression in neuroblastoma cells.

P2X7 receptor (P2RX7) is expressed strongly by most human cancers, including neuroblastoma, where high levels of P2RX7 are correlated with a poor prognosis for patients. Tonic activation of P2X7 receptor favors cell metabolism and angiogenesis, thereby promoting cancer cell proliferation, immunosuppression, and metastasis. Although understanding the mechanisms that control P2X7 receptor levels in neuroblastoma cells could be biologically and clinically relevant, the intracellular signaling pathways involved in this regulation remain poorly understood. Here we show that (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), an allosteric inhibitor of dual specificity phosphatases (DUSP) 1 and 6, enhances the expression of P2X7 receptor in N2a neuroblastoma cells. We found that exposure to BCI induces the phosphorylation of mitogen-activated protein kinases p38 and JNK, while it prevents the phosphorylation of ERK1/2. BCI enhanced dual specificity phosphatase 1 expression, whereas it induced a decrease in the dual specificity phosphatase 6 transcripts, suggesting that BCI-dependent inhibition of dual specificity phosphatase 1 may be responsible for the increase in p38 and JNK phosphorylation. The weaker ERK phosphorylation induced by BCI was reversed by p38 inhibition, indicating that this MAPK is involved in the regulatory loop that dampens ERK activity. The PP2A phosphatase appears to be implicated in the p38-dependent dephosphorylation of ERK1/2. In addition, the PTEN phosphatase inhibition also prevented ERK1/2 dephosphorylation, probably through p38 downregulation. By contrast, inhibition of the p53 nuclear factor decreased ERK phosphorylation, probably enhancing the activity of p38. Finally, the inhibition of either p38 or Sp1-dependent transcription halved the increase in P2X7 receptor expression induced by BCI. Moreover, the combined inhibition of both p38 and Sp1 completely prevented the effect exerted by BCI. Together, our results indicate that dual specificity phosphatase 1 acts as a novel negative regulator of P2X7 receptor expression in neuroblastoma cells due to the downregulation of the p38 pathway.

Epigenetic modulation of neuroblastoma enhances T cell and NK cell immunogenicity by inducing a tumor-cell lineage switch

BackgroundImmunotherapy in high-risk neuroblastoma (HR-NBL) does not live up to its full potential due to inadequate (adaptive) immune engagement caused by the extensive immunomodulatory capacity of HR-NBL. We aimed to...

Cathepsin L promotes chemresistance to neuroblastoma by modulating serglycin

Cathepsin L (CTSL), a lysosomal acid cysteine protease, is found to play a critical role in chemosencitivity and tumor progression. However, the potential roles and molecular mechanisms of CTSL in chemoresistance in neuroblastoma (NB) are still unclear. In this study, the correlation between clinical characteristics, survival and CTSL expression were assessed in Versteeg dataset. The chemoresistant to cisplatin or doxorubicin was detected using CCK-8 assay. Western blot was employed to detect the expression of CTSL, multi-drug resistance proteins, autophagy-related proteins and apoptosis-related proteins in NB cells while knocking down CTSL. Lysosome staining was analyzed to access the expression levels of lysosomes in NB cells. The expression of apoptosis markers was analyzed with immunofluorescence. Various datasets were analyzed to find the potential protein related to CTSL. In addition, a subcutaneous tumor xenografts model in M-NSG mice was used to assess tumor response to CTSL inhibition in vivo. Based on the validation dataset (Versteeg), we confirmed that CTSL served as a prognostic marker for poor clinical outcome in NB patients. We further found that the expression level of CTSL was higher in SK-N-BE (2) cells than in IMR-32 cells. Knocking down CTSL reversed the chemoresistance in SK-N-BE (2) cells. Furthermore, combination of CTSL inhibition and chemotherapy potently blocked tumor growth in vivo. Mechanistically, CTSL promoted chemoresistance in NB cells by up-regulating multi-drug resistance protein ABCB1 and ABCG2, inhibiting the autophagy level and cell apoptpsis. Furthermore, we observed six datasets and found that Serglycin (SRGN) expression was positively associated with CTSL expresssion. CTSL could mediate chemoresistance by up-regulating SRGN expression in NB cells and SRGN expression was positively correlated with poor prognosis of NB patients. Taken together, our findings indicate that the CTSL promotes chemoresistance to cisplatin and doxorubicin by up-regulating the expression of multi-drug resistance proteins and inhibiting the autophagy level and cell apoptosis in NB cells. Thus, CTSL may be a therapeutic target for overcoming chemoresistant to cisplatin and doxorubicin in NB patients.

CD24 Expression Dampens the Basal Antiviral State in Human Neuroblastoma Cells and Enhances Permissivity to Zika Virus Infection

Zika virus (ZIKV) exhibits distinct selectivity for infection of various cells and tissues, but how host cellular factors modulate varying permissivity remains largely unknown. Previous studies showed that the neuroblastoma cell line SK-N-AS (expressing low levels of cellular protein CD24) was highly restricted for ZIKV infection, and that this restriction was relieved by ectopic expression of CD24. We tested the hypothesis that CD24 expression allowed ZIKV replication by suppression of the antiviral response. SK-N-AS cells expressing an empty vector (termed CD24-low cells) showed elevated basal levels of phosphorylated STAT1, IRF-1, IKKE, and NFκB. In response to exogenously added type I interferon (IFN-I), CD24-low cells had higher-level induction of antiviral genes and activity against two IFN-I-sensitive viruses (VSV and PIV5-P/V) compared to SK-N-AS cells with ectopic CD24 expression (termed CD24-high cells). Media-transfer experiments showed that the inherent antiviral state of CD24-low cells was not dependent on a secreted factor such as IFN-I. Transcriptomics analysis revealed that CD24 expression decreased expression of genes involved in intracellular antiviral pathways, including IFN-I, NFκB, and Ras. Our findings that CD24 expression in neuroblastoma cells represses intracellular antiviral pathways support the proposal that CD24 may represent a novel biomarker in cancer cells for susceptibility to oncolytic viruses.

SNHG25 facilitates SNORA50C accumulation to stabilize HDAC1 in neuroblastoma cells

Increasing studies have pointed out that small nucleolar RNAs (snoRNAs) and their host genes (SNHGs) have multi-functional roles in cancer progression. Bioinformatics analysis revealed the importance of snoRNA host gene 25 (SNHG25) in neuroblastoma (NB). Hence, we further explored the function and molecular mechanism of SNHG25 in NB. Our study revealed that SNHG25 expression was upregulated in NB cells. Through loss-of-function assays, we discovered that silencing of SNHG25 suppressed NB cell proliferation, invasion, and migration. Moreover, we found that SNHG25 positively regulated snoRNA small nucleolar RNA, H/ACA box 50 C (SNORA50C) in NB cells, and SNORA50C depletion had the same function as SNHG25 silencing in NB cells. Moreover, we proved that SNHG25 recruited dyskerin pseudouridine synthase 1 (DKC1) to facilitate SNORA50C accumulation and associated small nucleolar ribonucleoprotein (snoRNP) assembly. In addition, it was manifested that SNHG25 relied on SNORA50C to inhibit ubiquitination of histone deacetylase 1 (HDAC1), thereby elevating HDAC1 expression in NB cells. Further, HDAC1 was proven to be a tumor-facilitator in NB, and SNORA50C contributed to NB cell growth and migration through the HDAC1-mediated pathway. In vivo xenograft experiments further supported that SNHG25 promoted NB progression through SNORA50C/HDAC1 pathway. Our study might provide a novel sight for NB treatment.

Characterizing the influence of Toxoplasma infection on opioid receptor expression using human neuroblastoma cells (SKNSH)

BackgroundToxoplasma gondii is an obligate intracellular parasite that has permanently infected billions of people. The parasite can convert to a latent form that is found preferentially in the brain and heart. Recent serological studies have linked Toxoplasma infection to many brain disorders including schizophrenia, depression, substance abuse and Alzheimer’s. However, the molecular mechanism by which Toxoplasma influences these brain disorders remains unclear. Previous studies have shown that Toxoplasmaincreases dopamine synthesis, and dopamine is an important neurotransmitter in the reward pathway, where the opioid system has been shown to play a major role in modulating this pathway. We aim to further explore these mechanisms by studying the influence of Toxoplasmainfection on opioid receptor expression.ObjectivesThe goal of our project is to elucidate the influence of Toxoplasma gondiion brain physiology through studying the impact of Toxoplasma infection on opioid receptor expression. The aim of this study is to determine the effect of Toxoplasma infection on opioid receptor expression in neuroblastoma cells (SKNSH) in the presence or absence of morphine.MethodsTo evaluate the potency of morphine against Toxoplasma, a doubling assay was performed. Briefly, Human foreskin fibroblasts (HFF) were infected with Toxoplasma. Six‐hours post‐infection, the uninvaded parasites were removed and the media was replaced with media containing 10 µM morphine, 10 µM pyrimethamine, or DMSO. The flasks were fixed with ice‐cold methanol and the number of parasites per vacuole were enumerated at 24‐ and 48‐hours post‐treatment. To evaluate the expression of opioid receptors, the neuroblastoma cell SKNSH, which has been shown to express functional mu and delta opioid receptors, was infected with Toxoplasma type 1. Four hours post‐infection, uninvaded parasites were removed and the medium was replaced with media containing DMSO or 1 µM morphine or 10 µM morphine. Thirty‐two hours post‐treatment, the cells were harvested and protein was isolated. The expression of mu and delta opioid receptors was evaluated using western blot analysis.ResultsWe observed changes in the morphology of SKNSH upon infection with Toxoplasma type 1. The results from our doubling assay demonstrate the lack of potency for morphine against Toxoplasma type 1 at 24 hours and 48 hours post treatment with the highest morphine concentration we have been using for these experiments. Preliminary results suggest that the expression of the delta opioid receptor is downregulated in the presence of Toxoplasma, independent of morphine administration. In addition, the expression of delta opioid receptor is increased in the presence of morphine alone. When pyrimethamine was added to cells containing both Toxoplasma and morphine, delta receptor expression was comparable to its expression in the presence of morphine alone.ConclusionWe have confirmed the expression of muand delta opioid receptors in SKNSH, as reported previously. In addition, we were able to successfully infect this cell line with Toxoplasma. Additionally, we have found that morphine lacks potency against Toxoplasma, unlike previously reported. Preliminary data suggest that the collective the presence of Toxoplasma can negatively influence the expression of delta opioid receptors. Future experiments are being designed to elucidate the molecular mechanism of these findings, as well as observing similar findings in the animal model of Toxoplasmosis.

Silencing of long chain noncoding RNA paternally expressed gene (PEG10) inhibits the progression of neuroblastoma by regulating microRNA-449a (miR-449a)/ribosomal protein S2 (RPS2) axis

ABSTRACT To investigate the mechanism of paternally expressed gene (PEG10) in regulating neuroblastoma (NB) progression. PEG10 expression was detected using quantitative real-time reverse transcription polymerase-chain reaction (qRT-PCR). The interaction of miR-449a and PEG10 or ribosomal protein S2 (RPS2) was employed by starBase, and then proved through RIP and dual-luciferase reporter assays. The NB cell viability, proliferation, invasion, and migration were evaluated by Cell Counting Kit-8 (CCK-8), colony formation, and Transwell assay. The mRNA and protein levels were determined by qRT-PCR and Western blotting, respectively. The levels of PEG10 and RPS2 were remarkably increased in NB tissues and cells, nevertheless the expression of miR-449a was conspicuously declined in NB tissues and cells. Silencing of PEG10 inhibited proliferation, migration, and invasion in SK-N-BE (2) cells, while overexpression of PEG10 promoted proliferation, migration, and invasion in SH-SY5Y cells. We affirmed that PEG10 interacted with miR-449a, and miR-449a could target the 3ʹUTR of RPS2 and negatively regulate its expression in NB cells. The upregulation of miR-449a inhibited proliferation, migration, and invasion in SK-N-BE (2) cells, while downregulation of miR-449a promoted proliferation, migration, and invasion in SH-SY5Y cells. Moreover, miR-449a overexpression weaken the function of PEG10-mediated on promoting proliferation, migration, and invasion in SH-SY5Y cells, while RPS2 overexpression rescued the effects of miR-449a-mediated on inhibiting those behaviors of SH-SY5Y cells. In conclusion, Silencing of PEG10 could inhibit proliferation, migration, and invasion via the miR-449a/RPS2 axis in NB cells.

Identification of Mitochondrial DNA Variants Associated With Risk of Neuroblastoma.

Neuroblastoma is a childhood cancer that originates in the developing sympathetic nervous system. We previously reported a crucial role of mitochondrial DNA haplogroups in the pathology of neuroblastoma. To pinpoint mitochondrial DNA variants associated with neuroblastoma risk, we applied a mitochondrial genome imputation pipeline to the single nucleotide polymorphisms array data of 2 pediatric cohorts containing a total of 2404 neuroblastoma patients and 9310 cancer-free controls. All statistical tests were 2-sided. The single nucleotide variant, rs2853493, was statistically significantly associated with neuroblastoma risk in the discovery cohort (odds ratio = 0.62, 95% confidence interval = 0.53 to 0.72, P < .001) and further confirmed in the replication cohort (odds ratio = 0.75, 95% confidence interval = 0.62 to 0.90, P = .002). Further, expression quantitative trait loci analysis indicated genotypes of rs2853493 were associated with expression levels of MT-CYB gene expression in neuroblastoma cells, suggesting rs2853493 may confer risk to neuroblastoma via regulating the expression level of its nearby genes.

CircRNA-ACAP2 contributes to the invasion, migration, and anti-apoptosis of neuroblastoma cells through targeting the miRNA-143-3p-hexokinase 2 axis.

BackgroundCirculating RNAs (Circ-RNAs) are tightly related to the processes of neuroblastoma. The circ-ACAP2 has been reported as dysregulated in various cancers; however, its biological roles and mechanisms in neuroblastoma remain largely unclear.MethodsWe collected 40 neuroblastoma tissues and adjacent noncancerous tissues. Quantitative reverse transcription polymerase chain reaction (qRT-RCR) or western blot were used to examine ACAP2, miR-143-3p, and HK2 abundances. Cell migration, invasion, glycolysis, and apoptosis were assessed via wound healing, transwell, glucose uptake and lactate, 3-(4,5-diamethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and flow cytometry. The association between circRNA, microRNA (miRNA), and messenger RNA (mRNA) was examined by dual-luciferase reporter analysis and RNA immunoprecipitation.ResultsThe abundances of ACAP2 and HK2 were remarkedly increased in neuroblastoma tissues and cell lines. Silencing ACAP2 significantly constrained neuroblastoma cell migration, invasion, and glycolysis, and promoted apoptosis. Bioinformatics prediction, luciferase assay, and RNA pull-down assay consistently demonstrated that ACAP2 sponged miR-143-3p to downregulate its expression in neuroblastoma cells. Furthermore, we identified that hexokinase 2, a glycolysis key enzyme, was a direct target of miR-143-3p in neuroblastoma cells. Rescue of miR-143-3p in ACAP2-overexpressing cells effectively mitigated the influence of ACAP2 on neuroblastoma cell processes.ConclusionsOur study revealed biological roles and molecular mechanisms for circ-ACAP2 in the oncogenic characteristics of neuroblastoma, facilitating the development of circRNA-based treatment approaches for anti-brain tumor therapy.

BORIS/CTCFL expression activates the TGFβ signaling cascade and induces Drp1 mediated mitochondrial fission in neuroblastoma

The cancer-testis antigen CTCFL/BORIS (Brother of Regulator of Imprinted Sites) also known, as a paralog of CTCF -the “master weaver of the genome” is a key transcriptional regulator. Both CTCF and BORIS can bind to the same promoter sequence and recruit diverse proteins. BORIS is also known to be associated with actively translating ribosomes suggesting new roles of BORIS in gene expression. Various studies have attempted to elucidate the role of BORIS in different cell types for the development of targeted therapy depending on molecular signatures and genetic aberrations associated with the disease type. The current study is focused on its role in neuroblastoma. Here, we have deciphered the role of BORIS on TGFβ1 pathway which is highly affected by embryonic CTCFL expression. BORIS stabilized the SMAD3 and SMAD4 transcripts leading to prolonged TGFβ activation. Further, loss of BORIS abrogated both the canonical and non-canonical TGFβ signaling suggesting the dependency of TGFβ on BORIS. The effect on the metabolic profile of the neuroblastoma cells were analyzed with change in BORIS expression levels. Also, ectopic expression of BORIS leads to Drp1 phosphorylation (Ser616) enhancing mitochondrial fission followed by a switch in cellular metabolism towards glycolysis. This cellular metabolism switch was in turn supported with a reduction in oxygen consumption rate upon BORIS expression. Interestingly methylome analysis revealed patterns of global histone methylation, a mechanism that regulate important signaling pathways in neuroblastoma. This study analyzes the consequence of BORIS expression in neuroblastoma cells and thereby elucidate its downstream targets, which could help in designing effective therapeutic for treating neuroblastoma. Similar results were obtained in both MYCN amplified and non-MYCN neuroblastoma cell lines, indicating a common mechanism of BORIS/CTCFL action in neuroblastoma.

Adipose-derived stem cell-derived extracellular vesicles inhibit neuroblastoma growth by regulating GABBR1 activity through LINC00622-mediated transcription factor AR.

Neuroblastoma (NB) is a huge threat to children's health. Adipose-derived stem cells-derived extracellular vesicles (ADSC-Evs) can regulate tumor progression. This study aimed to identify the role of ADSC-Evs in NB. Following ADSC-Ev isolation and identification, PKH26-labeled ADSC-Evs were cocultured with NB cells to observe the internalization of ADSC-Evs. ADSC-Ev effects on NB cell proliferation, invasion, and migration were assessed. The regulatory molecules related to NB development were predicted. The expressions of and relations among LINC00622, transcriptional factor androgen receptor (AR), and gamma-aminobutyric acid B-type receptor 1 (GABRR1) were detected and verified. LINC00622 was inhibited in ADSCs to evaluate ADSC-Ev effects on NB cells. Xenograft tumor experiment in nude mice was further performed to evaluate the effects of ADSC-Evs-carried LINC00622 on NB in vivo. ADSC-Evs inhibited NB cell proliferation, invasion, and migration. ADSC-Evs increased GABBR1 expression in NB cells. ADSC-Evs-carried LINC00622 mediated AR to promote GABBR1 expression. Silencing LINC00622 in ADSCs weakened the inhibition of ADSC-Evs on NB cell malignant behaviors. ADSC-Evs reduced tumor growth in nude mice, which was restored after inhibiting LINC00622 expression in ADSCs. We highlighted that ADSC-Evs carried LINC00622 into NB cells to inhibit transcription factor AR and promote GABBR1 expression, thus inhibiting NB cell growth.

CD47-SIRPα Checkpoint Inhibition Enhances Neutrophil-Mediated Killing of Dinutuximab-Opsonized Neuroblastoma Cells.

Simple SummaryCurrent immunotherapy for high-risk neuroblastoma patients involves treatment with anti-GD2 antibody dinutuximab, which has significantly improved the survival rate. Still, approximately half of the patients succumb to the tumor; therefore, efforts to improve their prognosis are urgently needed. Since T cell targeting immune checkpoint inhibitors in neuroblastoma are limited due to the low immunogenicity of these tumors, alternative immunotherapeutic approaches should be studied. The therapeutic targeting of the innate immune checkpoint CD47-SIRPα has the ability to enhance antitumor effects of myeloid cells, especially in the presence of cancer-opsonizing antibodies. Given that neutrophil ADCC is a dominant effector mechanism leading to the eradication of dinutuximab-opsonized neuroblastoma cells, we have investigated the therapeutic potential of anti-GD2 antibody in combination with CD47-SIRPα inhibition. We demonstrate here that the capacity of neutrophils to kill dinutuximab-opsonized neuroblastoma cells is controlled by the CD47-SIRPα axis and its disruption promotes their cytotoxic potential even further, significantly improving dinutuximab responsiveness.High-risk neuroblastoma, especially after recurrence, still has a very low survival rate. Immune checkpoint inhibitors targeting T cells have shown remarkable clinical efficacy in adult solid tumors, but their effects in pediatric cancers have been limited so far. On the other hand, targeting myeloid immune checkpoints, such as CD47-SIPRα, provide the opportunity to enhance antitumor effects of myeloid cells, including that of neutrophils, especially in the presence of cancer-opsonizing antibodies. Disialoganglioside (GD2)-expressing neuroblastoma cells targeted with anti-GD2 antibody dinutuximab are in part eradicated by neutrophils, as they recognize and bind the antibody targeted tumor cells through their Fc receptors. Therapeutic targeting of the innate immune checkpoint CD47-SIRPα has been shown to promote the potential of neutrophils as cytotoxic cells in different solid tumor indications using different cancer-targeting antibodies. Here, we demonstrate that the capacity of neutrophils to kill dinutuximab-opsonized neuroblastoma cells is also controlled by the CD47-SIRPα axis and can be further enhanced by antagonizing CD47-SIRPα interactions. In particular, CD47-SIRPa checkpoint inhibition enhanced neutrophil-mediated ADCC of dinutuximab-opsonized adrenergic neuroblastoma cells, whereas mesenchymal neuroblastoma cells may evade immune recognition by a reduction of GD2 expression. These findings provide a rational basis for targeting CD47-SIRPα interactions to potentiate dinutuximab responsiveness in neuroblastomas with adrenergic phenotype.