Neuroblastoma Cell Proliferation Research Articles

YAP promotes the proliferation of neuroblastoma cells through decreasing the nuclear location of p27Kip1 mediated by Akt.

ObjectiveWe aimed to investigate the roles and underlying mechanisms of YAP in the proliferation of neuroblastoma cells.MethodsThe expression level of YAP was evaluated by Western blotting and immunocytochemistry. Cell viability, cell proliferation and growth were detected by CCK‐8, PH3 and Ki67 immunostaining, and the real‐time cell analyser system. The nuclear and cytoplasmic proteins of p27Kip1 were dissociated by the nuclear‐cytosol extraction kit and were detected by Western blotting and immunocytochemistry. mRNA levels of Akt, CDK5 and CRM1 were determined by qRT‐PCR.ResultsYAP was enriched in SH‐SY5Y cells (a human neuroblastoma cell line). Knock‐down of YAP in SH‐SY5Y cells or SK‐N‐SH cell line (another human neuroblastoma cell line) significantly decreased cell viability, inhibited cell proliferation and growth. Mechanistically, knock‐down of YAP increased the nuclear location of p27Kip1, whereas serum‐induced YAP activation decreased the nuclear location of p27Kip1 and was required for cell proliferation. Meanwhile, overexpression of YAP in these serum‐starved SH‐SY5Y cells decreased the nuclear location of p27Kip1, promoted cell proliferation and overexpression of p27Kip1 in YAP‐activated cells inhibited cell proliferation. Furthermore, knock‐down of YAP reduced Akt mRNA and protein levels. Overexpression of Akt in YAP‐downregulated cells decreased the nuclear location of p27Kip1 and accelerated the proliferation of SH‐SY5Y cells.ConclusionsOur studies suggest that YAP promotes the proliferation of neuroblastoma cells through negatively controlling the nuclear location of p27Kip1 mediated by Akt.

Telomerase Is a Prognostic Marker of Poor Outcome and a Therapeutic Target in Neuroblastoma.

Telomere maintenance is a hallmark of high-risk neuroblastoma; however, the contribution of telomerase and alternative lengthening of telomeres (ALT) to clinical phenotypes has remained unclear. We aimed to determine the clinical relevance of telomerase activation versus ALT as biomarkers in pretreatment neuroblastoma and to assess the potential value of telomerase as a therapeutic target. The genomic status of TERT and MYCN was assessed in 457 pretreatment neuroblastomas by fluorescence in situ hybridization. ALT was examined in 273 of 457 tumors by detection of ALT-associated promyelocytic leukemia nuclear bodies, and TERT expression was determined by microarrays in 223 of these. Cytotoxic effects of telomerase-interacting compounds were analyzed in neuroblastoma cell lines in vitro and in vivo. We detected TERT rearrangements in 46 of 457 cases (10.1%), MYCN amplification in 93 of 457 cases (20.4%), and elevated TERT expression in tumors lacking TERT or MYCN alterations in 10 of 223 cases (4.5%). ALT activation was found in 49 of 273 cases (17.9%). All these alterations occurred almost mutually exclusively and were associated with unfavorable prognostic variables and adverse outcome. The presence of activated telomerase (ie, TERT rearrangements, MYCN amplification, or high TERT expression without these alterations) was associated with poorest overall survival and was an independent prognostic marker in multivariable analyses. We also found that the telomerase-interacting compound 6-thio-2'-deoxyguanosine effectively inhibited viability and proliferation of neuroblastoma cells bearing activated telomerase. Similarly, tumor growth was strongly impaired upon 6-thio-2'-deoxyguanosine treatment in telomerase-positive neuroblastoma xenografts in mice. Our data suggest telomerase activation and ALT define distinct neuroblastoma subgroups with adverse outcome and that telomerase may represent a promising therapeutic target in many high-risk neuroblastomas.

Abstract A101: Nucleolin: A novel cell surface protein for neuroblastoma targeted therapy

Abstract Background: Neuroblastoma (NB) represents the most frequent and aggressive form of extracranial solid tumor of infants. Since the use of targeted therapies is severely limited by the lack of specific receptors, chemotherapy and radiotherapy remain the main treatment choices, which, nevertheless expose high-risk NB patients to unacceptable toxicity and to the development of drug resistance. Nucleolin (NCL), a multifunctional protein involved in both physiological and pathological conditions, is overexpressed and partially localized on the cell surface of tumor cells of adult cancers. Little is reported about NCL in pediatric tumors, including NB. Methods: To test the expression of NCL as a potential and targetable cell surface marker in NB, human NB cell lines were evaluated in vitro and after injection in mice, by Imaging Flow Cytometry (IFC) and immunohistochemistry (IHC) analyses, respectively. Cell binding and internalization of rhodamine-labeled nanocarriers, decorated with the NCL-recognizing F3 peptide (Rhod-F3-SL) were evaluated in vitro by FACS and confocal microscopy. The cytotoxic potential of a doxorubicin (DXR)-loaded, pH-sensitive nanocarrier and decorated with the F3 peptide was evaluated in vitro, in terms of inhibition of NB cells proliferation and induction of cell death by CSFE and MTS assays, respectively. Orthotopic and pseudometastatic animal models of human NB, mimicking the growth and spread of NB in patients, were finally enrolled to examine in vivo the anti-tumor activity of DXR-loaded, pH-sensitive nanocarriers decorated with F3 peptide. Results: IFC analyses demonstrated that NCL is expressed at different extent on the surface of all NB cells analyzed. Notably, IHC evaluations displayed NCL staining on both tumor and endothelial tumor cells in well-established NB xenografts. Moreover, in vitro binding studies showed that Rhod-F3-SL strongly associated to NB cells, exhibiting a specific co-localization with cell surface NCL. The cellular distribution of Rhod-F3-SL also confirms the binding specificity, showing an increased selective internalization of NCL-targeted nanoparticles, compared to that obtained with untargeted formulations. Finally, F3-targeted, DXR-loaded, pH-sensitive nanocarriers resulted significantly more effective, in terms of inhibition of cell proliferation and reduction of cell viability in vitro, and in terms of delay of tumor growth in each NB animal model tested, when compared to DXR-loaded, pH-sensitive untargeted liposomes. Conclusion: Our findings demonstrate that NCL represents a novel cell surface protein to be targeted by “drug”-loaded nanoparticles as an innovative therapeutic strategy for high-risk NB. Citation Format: Brignole Chiara, Veronica Bensa, Genny Del Zotto, Silvia Bruno, Nuno A Fonseca, Ana F Cruz, Daniela Di Paolo, Patrizia Perri, Vera Moura, Laura Emionite, Michele Cilli, Mirco Ponzoni, Joao N Moreira, Fabio Pastorino. Nucleolin: A novel cell surface protein for neuroblastoma targeted therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A101. doi:10.1158/1535-7163.TARG-19-A101

MiR-15a-5p, miR-15b-5p, and miR-16-5p inhibit tumor progression by directly targeting MYCN in neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid malignancy in children. Despite current aggressive treatment regimens, the prognosis for high‐risk NB patients remains poor, with the survival of less than 40%. Amplification/stabilization of MYCN oncogene, in NB is associated with a high risk of recurrence. Thus, there is an urgent need for novel therapeutics. The deregulated expression of microRNA (miR) is reported in NB; nonetheless, its effect on MYCN regulation is poorly understood. First, we identified that miR‐15a‐5p, miR‐15b‐5p, and miR‐16‐5p (hereafter miR‐15a, miR‐15b or miR‐16) were down‐regulated in patient‐derived xenografts (PDX) with high MYCN expression. MiR targeting sequences on MYCN mRNA were predicted using online databases such as TargetScan and miR database. The R2 database, containing 105 NB patients, showed an inverse correlation between MYCN mRNA and deleted in lymphocytic leukemia (DLEU) 2, a host gene of miR‐15. Moreover, overexpression of miR‐15a, miR‐15b or miR‐16 significantly reduced the levels of MYCN mRNA and N‐Myc protein. Conversely, inhibiting miR dramatically enhanced MYCN mRNA and N‐Myc protein levels, as well as increasing mRNA half‐life in NB cells. By performing immunoprecipitation assays of argonaute‐2 (Ago2), a core component of the RNA‐induced silencing complex, we showed that miR‐15a, miR‐15b and miR‐16 interact with MYCN mRNA. Luciferase reporter assays showed that miR‐15a, miR‐15b and miR‐16 bind with 3’UTR of MYCN mRNA, resulting in MYCN suppression. Moreover, induced expression of miR‐15a, miR‐15b and miR‐16 significantly reduced the proliferation, migration, and invasion of NB cells. Finally, transplanting miR‐15a‐, miR‐15b‐ and miR‐16‐expressing NB cells into NSG mice repressed tumor formation and MYCN expression. These data suggest that miR‐15a, miR‐15b and miR‐16 exert a tumor‐suppressive function in NB by targeting MYCN. Therefore, these miRs could be considered as potential targets for NB treatment.

Inhibiting effect of oleocanthal on neuroblastoma cancer cell proliferation in culture

ABSTRACTWe investigated the potential anticancer effects of oleocanthal (OC) on neuroblastoma cells. Cells were divided into four groups: group 1, neuroblastoma cells were treated with OC; group 2, neurons that differentiated from neuroblastoma cells were treated with phosphate-buffered saline(PBS); group 3, bone marrow derived neuronal (BMDN) cells that were differentiated from bone marrow derived mesenchymal stem cells (BMSCs) were treated with OC; group 4, BMDN cells that were differentiated from BMSCs were treated with PBS. Groups 2 and 4 were control groups. The effects of OC on cell viability, oxidative stress, neurite inhibition and apoptosis at IC50 dose were investigated using MTT analysis, i-NOS and e-NOS measurement, neurotoxicity screening test (NST) and TUNEL staining, respectively. MTT analysis demonstrated that cells were significantly less viable in group 1 than in group 3. i-NOS and e-NOS staining intensity was significantly greater in group 1 than in group 3. NST revealed that OC inhibited neurite growth in both neuroblastoma and BMND cells; inhibition was significantly less in group 3 than in group 1. Significantly more TUNEL labeled cells were found in group 1 than in group 3. We found that OC prevented growth and proliferation of neuroblastoma cells in culture by increasing oxidative stress and apoptosis. We also found that the cytotoxicity of OC is negligible in BMDN cells.

Silencing CASC11 curbs neonatal neuroblastoma progression through modulating microRNA-676-3p/nucleolar protein 4 like (NOL4L) axis.

Neuroblastoma is the commonest extracranial solid cancer for neonates. Long non-coding RNA cancer susceptibility 11 (CASC11) is corroborated as carcinogen in several tumors. But its role in neonatal neuroblastoma is poorly defined. Expression levels of CASC11, miR-676-3p, and NOL4L mRNA were analyzed by qRT-PCR in cells and tissues. Kaplan-Meier analysis was used to measure and analyze the survival time of patients with high/low CASC11. Neonatal neuroblastoma cell proliferation was reflected through colony-formation assay and CCK-8. Transwell assay was designed for detection of migratory and invasive capacities of neonatal neuroblastoma cells. Wound-healing assay was used for monitoring neuroblastoma cell migration. RNA pull-down, luciferase reporter, and RIP assays were utilized to identify the relationship between CASC11, miR-676-3p, and NOL4L on the basis of bioinformatics tools. Highly expressed CASC11 was observed in neonatal neuroblastoma tissues and cells. High level of CASC11 indicated unsatisfactory survival of neonatal neuroblastoma patients. CASC11 depletion inhibited cell proliferation and invasiveness. CASC11 was a molecular sponge to release NOL4L from miR-676-3p inhibition in tumor cells. Upregulation of NOL4L abated the suppressed cell proliferation and invasiveness due to CASC11 downregulation. CASC11 sequestered miR-676-3p from NOL4L to facilitate neonatal neuroblastoma progression, hinting a CASC11-mediated therapeutic target for neonatal neuroblastoma.

CDGSH Iron Sulfur Domain 2 Deficiency Inhibits Cell Proliferation and Induces Cell Differentiation of Neuroblastoma.

CDGSH iron sulfur domain 2 (CISD2) is reported to be highly expressed in several cancers, but the role of it in neuroblastoma has not been identified yet. Here, for the first time, we show that CISD2 is involved in neuroblastoma tumorigenesis and regulates neuroblastoma cell proliferation and differentiation. We found that high CISD2 expression correlated significantly with poor outcome of neuroblastoma patients, as well as advanced neuroblastoma tumor stages. Knockdown of CISD2 greatly repressed neuroblastoma cell proliferation and tumorigenesis both in vitro and in vivo. Further investigation showed that CISD2 deficiency resulted in cell cycle arrest in G1 phase and induced cell differentiation of neuroblastoma. Several Cyclins and Cyclin-Dependent Kinases (CDKs) were down-regulated by CISD2 knockdown, indicating that CISD2 probably regulates cell cycle through those genes. Together, we provide evidence that CISD2 is an indicator for neuroblastoma patients prognosis and is indispensable for neuroblastoma cell proliferation and tumorigenesis; CISD2 deficiency can induce neuroblastoma cell cycle arrest and differentiation. These findings suggest that CISD2 could work as a novel and potential therapeutic target for neuroblastoma treatment.

Combinatorial targeting of MTHFD2 and PAICS in purine synthesis as a novel therapeutic strategy

MYCN-amplified (MNA) neuroblastoma is an aggressive neural crest-derived pediatric cancer. However, MYCN is indispensable for development and transcriptionally regulates extensive network of genes. Integrating anti-MYCN ChIP-seq and gene expression profiles of neuroblastoma patients revealed the metabolic enzymes, MTHFD2 and PAICS, required for one-carbon metabolism and purine biosynthesis were concomitantly upregulated, which were more susceptible to metastatic neuroblastoma. Moreover, we found that MYCN mediated the folate cycle via MTHFD2, which contributed one-carbon unit to enhance purine synthesis, and further regulated nucleotide production by PAICS in response to cancer progression. Dual knockdown of the MYCN-targeted gene pair, MTHFD2 and PAICS, in MNA neuroblastoma cells synergically reduced cell proliferation, colony formation, migration ability, and DNA synthesis. By systematically screening the compound perturbagens, the gene expression levels of MTHFD2 and PAICS were specifically suppressed by anisomycin and apicidin across cell lines, and our co-treatment results also displayed synergistic inhibition of MNA neuroblastoma cell proliferation. Collectively, targeting a combination of MYCN-targeted genes that interrupts the interconnection of metabolic pathways may overcome drug toxicity and improve the efficacy of current therapeutic agents in MNA neuroblastoma.

HMGB1 Receptors as Potential Novel Targets for Neuroblastoma

Background and Hypothesis: The pathogenesis of neuroblastoma remains elusive. In order to further understand the pathogenesis, it is necessary to identify molecular and cellular mechanisms of the cancer. One protein of interest is high-mobility group box 1 protein (HMGB1). HMGB1 has been implicated in cancer including tumor growth, invasion, angiogenesis, metastasis, relapse and therapeutic resistance. HMGB1 isoforms signals via different receptors including the chemokine receptor, CXCR4, receptor for advanced glycation end products (RAGE), and the prominent inflammation-associated receptor, toll-like receptor 4 (TLR4). To gain insight into the possible impact of HMGB1 on the SH-SY5Y neuroblastoma cell line, we determined the degree to which CXCR4, TLR4 and RAGE are present on SH-SY5Y cells. We hypothesize that the presence of these receptors in SH-SY5Y cells may mediate proliferation of neuroblastoma cells and other types of cancer.
 Experimental Design or Project Methods: The SH-SY5Y cell line (ATCC® CRL-2266™) was derived from a metastatic bone tumor in a 4 year old female. Cells were lysed in lysis buffer, electrophoresed on a 10% SDS-PAGE, and blotted onto PVDF membrane. After blocking, the membranes were incubated with primary antibodies against the receptor protein overnight at 4 °C, and then with HRP-conjugated secondary antibodies for 1h. Protein bands were visualized with a SuperSignal West Pico Chemiluminescent Substrate.
 Results: To be finalized 
 Conclusion and Potential Impact: Investigating SH-SY5Y cell line for the presence of these inflammation-associated receptors could potentially serve as a model to better understand the role of these receptors in cancer research.

Downregulation of microRNA‑3934‑5p induces apoptosis and inhibits the proliferation of neuroblastoma cells by targeting TP53INP1

Neuroblastoma is the most common pediatric extracranial solid tumour in the world. miRNAs are a group of endogenous small non-coding RNAs that act on target genes to serve critical roles in many biological processes. Presently, the expression and role of miR-3934-5p in neuroblastoma remains unclear. Therefore, the aim of the present study was to investigate the expression of miR-3934-5p in neuroblastoma tissues and cell lines and to assess the role of miR-3934-5p in neuroblastoma. In the current study, the results revealed that miR-3934-5p was significantly upregulated in neuroblastoma tissues and cell lines. The data also identified TP53INP1 as a direct target gene of miR-3934-5p, which was negatively regulated by miR-3934-5p. The present study further demonstrated that TP53INP1 was downregulated in both neuroblastoma tissues and cell lines. Furthermore, the results of the current study indicate that miR-3934-5p downregulation may induce apoptosis and inhibit neuroblastoma cell viability. However, these effects were reversed via TP53INP1-siRNA. Data from the current study indicates that the miR-3934-5p/TP53INP1 axis may be a novel therapeutic target for neuroblastoma treatment.

Ginsenoside Compound K Induces Ros-Mediated Apoptosis and Autophagic Inhibition in Human Neuroblastoma Cells In Vitro and In Vivo

Autophagy can result in cellular adaptation, as well as cell survival or cell death. Modulation of autophagy is increasingly regarded as a promising cancer therapeutic approach. Ginsenoside compound K (CK), an active metabolite of ginsenosides isolated from Panax ginseng C.A. Meyer, has been identified to inhibit growth of cancer cell lines. However, the molecular mechanisms of CK effects on autophagy and neuroblastoma cell death have not yet been investigated. In the present study, CK inhibited neuroblastoma cell proliferation in vitro and in vivo. Treatment by CK also induced the accumulation of sub-G1 population, and caspase-dependent apoptosis in neuroblastoma cells. In addition, CK promotes autophagosome accumulation by inducing early-stage autophagy but inhibits autophagic flux by blocking of autophagosome and lysosome fusion, the step of late-stage autophagy. This effect of CK appears to be mediated through the induction of intracellular reactive oxygen species (ROS) and mitochondria membrane potential loss. Moreover, chloroquine, an autophagy flux inhibitor, further promoted CK-induced apoptosis, mitochondrial ROS induction, and mitochondria damage. Interestingly, those promoted phenomena were rescued by co-treatment with a ROS scavenging agent and an autophagy inducer. Taken together, our findings suggest that ginsenoside CK induced ROS-mediated apoptosis and autophagic flux inhibition, and the combination of CK with chloroquine, a pharmacological inhibitor of autophagy, may be a novel therapeutic potential for the treatment of neuroblastoma.

Glycine decarboxylase is a transcriptional target of MYCN required for neuroblastoma cell proliferation and tumorigenicity.

Genomic amplification of the oncogene MYCN is a major driver in the development of high-risk neuroblastoma, a pediatric cancer with poor prognosis. Given the challenge in targeting MYCN directly for therapy, we sought to identify MYCN-dependent metabolic vulnerabilities that can be targeted therapeutically. Here, we report that the gene encoding glycine decarboxylase (GLDC), which catalyzes the first and rate-limiting step in glycine breakdown with the production of the one-carbon unit 5,10-methylene-tetrahydrofolate, is a direct transcriptional target of MYCN. As a result, GLDC expression is markedly elevated in MYCN-amplified neuroblastoma tumors and cell lines. This transcriptional upregulation of GLDC expression is of functional significance, as GLDC depletion by RNA interference inhibits the proliferation and tumorigenicity of MYCN-amplified neuroblastoma cell lines by inducing G1 arrest. Metabolomic profiling reveals that GLDC knockdown disrupts purine and central carbon metabolism and reduces citrate production, leading to a decrease in the steady-state levels of cholesterol and fatty acids. Moreover, blocking purine or cholesterol synthesis recapitulates the growth inhibitory effect of GLDC knockdown. These findings reveal a critical role of GLDC in sustaining the proliferation of neuroblastoma cells with high-level GLDC expression and suggest that MYCN amplification is a biomarker for GLDC-based therapeutic strategies against high-risk neuroblastoma.

Down-Regulation of Phosphoribosyl Pyrophosphate Synthetase 1 Inhibits Neuroblastoma Cell Proliferation.

Phosphoribosyl pyrophosphate synthetase 1 (PRPS1) is a key enzyme in de novo nucleotide synthesis and nucleotide salvage synthesis pathways that are critical for purine and pyrimidine biosynthesis. Abnormally high expression of PRPS1 can cause many diseases, including hearing loss, hypotonia, and ataxia, in addition to being associated with neuroblastoma. However, the role of PRPS1 in neuroblastoma is still unclear. In this study, we found that PRPS1 was commonly expressed in neuroblastoma cells and was closely related to poor prognosis for cancer. Furthermore, down-regulation of PRPS1 inhibited neuroblastoma cell proliferation and tumor growth in vitro and in vivo via disturbing DNA synthesis. This study provides new insights into the treatment of neuroblastoma patients and new targets for drug development.

Identification of α-N-catenin as a novel tumor suppressor in neuroblastoma.

The lost expression of α-catenin has been found in cancers, and reinstalling α-catenin inhibits tumor growth. Here we hypothesized that the α-N-catenin, a homologous member of α-catenin and neural-specific expressed, functions as a novel tumor suppressor in neural crest-derived tumor, neuroblastoma. We correlated CTNNA2 (encodes α-N-catenin) expression to neuroblastoma disease relapse-free survival probability using publicly accessible human neuroblastoma datasets in R2 platform. The result showed that it negatively correlated to relapse-free survival probability significantly in patients with neuroblastoma with non-MYCN amplified tumor. Conversely, overexpressing CTNNA2 suppressed the neuroblastoma cell proliferation as measuring by the clonogenesis, inhibited anchorage-independent growth with soft agar colony formation assay. Forced expression of CTNNA2 decreased cell migration and invasion. Further, overexpression of CTNNA2 reduced the secretion of angiogenic factor IL-8 and HUVEC tubule formation. Our results show, for the first time, that α-N-catenin is a tumor suppressor in neuroblastoma cells. These findings were further corroborated with in vivo tumor xenograft study, in which α-N-catenin inhibited tumor growth and reduced tumor blood vessel formation. Interestingly, this is only observed in SK-N-AS xenografts lacking MYCN expression, and not in BE(2)-C xenografts with MYCN amplification. Mechanistically, α-N-catenin attenuated NF-κB responsive genes by inhibiting NF-κB transcriptional activity. In conclusion, these data demonstrate that α-N-catenin is a tumor suppressor in non-MYCN-amplified neuroblastomas and it inhibits NF-κB signaling pathway to suppress tumor growth in human neuroblastomas. Therefore, restoring the expression of α-N-catenin can be a novel therapeutic approach for neuroblastoma patients who have the deletion of CTNNA2 and lack of MYCN amplification.

β2‑adrenergic receptor signaling promotes neuroblastoma cell proliferation by activating autophagy.

Accumulating evidence suggests the pivotal role of the sympathetic nervous system in the initiation and aggressive progression of tumors, whereas the role of β-adrenergic receptor (β-AR) signaling in neuroblastoma (NB) and the underlying regulatory mechanisms have not yet been well elucidated. In the present study, it was demonstrated that the expression of both β1-AR and β2-AR was significantly increased in clinical samples of NB compared with those of ganglioneuroma (GN) and ganglioneuroblastoma (GNB), and that β2-AR is the key β-adrenergic receptor responsible for NB cell growth. Further investigation showed that the expression levels of the autophagy markers LC3-II, beclin-1 and unc-51-like autophagy kinase 1 (ULK1) were also elevated in NB, compared to the cases of GN and GNB. Moreover, β2-AR expression was found to be positively associated with autophagy markers in the clinical NB specimens. Cellular functional assays demonstrated that β2-AR activation promoted NB cell growth and activated the autophagy pathway. Pharmacological inhibition of autophagy with 3-methyladenine abolished β2-AR-induced NB cell growth. Mechanistically, β2-AR signaling triggers autophagy through CREB-mediated ULK1 upregulation. In conclusion, the present study uncovered a novel regulatory mechanism of β2-AR-activated autophagy in NB cell growth and provides a novel potential therapeutic approach for treating NB by targeting autophagy and the β2-AR pathway.

Linc01105 acts as an oncogene in the development of neuroblastoma

Previous research from our group revealed that the long coding RNA (lncRNA) linc01105 is associated with neuroblastoma proliferation and apoptosis, and that its expression is correlated with the International Neuroblastoma Staging System stage. The purpose of the present study was to investigate the functions of Linc01105 in neuroblastoma. Lentivirus‑mediated linc01105 knockdown was performed in the neuroblastoma cell line SH‑SY5Y. The expression levels of linc01105 and of other associated genes were measured by reverse transcription‑quantitative PCR. Cell Counting Kit‑8 assay and flow cytometry were used to determine cell viability and apoptosis. The levels of proteins were detected using western blot analysis. Bioinformatics analysis and luciferase reporter assays were used to examine the relationship between linc01105, miR‑6769b‑5p and vascular endothelial growth factor A (VEGFA). Angiogenesis ability was measured using a tube formation assay. The results demonstrated that HIF‑1α overexpression promoted the transcription of linc01105 by acting as a transcription factor. Knockdown of linc01105 inhibited neuroblastoma cell proliferation, migration and invasion, and it induced apoptosis. In addition, linc01105 affected the expression of p53 and Bcl‑2 family proteins and activated the caspase signaling pathway. Further functional experiments revealed that linc01105 promoted the expression of the miR‑6769b‑5p target gene VEGFA by acting as a sponge of miR‑6769b‑5p. In conclusion, linc01105 may contribute to neuroblastoma tumorigenesis and development. The present findings indicated that the interplay between the p53/caspase pathway and the linc01105/miR‑6769b‑5p/VEGFA axis may have important roles in the development of neuroblastoma.

MiR-34a inhibits proliferation, migration and invasion of paediatric neuroblastoma cells via targeting HNF4α

Objective To investigate the potential mechanism of microRNA-34a (miR-34a) on proliferation, migration and invasion of paediatric neuroblastoma cells. Methods The expression of miR-34a and hepatocyte nuclear factor 4α (HNF4α) in paediatric neuroblastoma tissues were detected by RT-q PCR and Western blot, respectively. Cell proliferation, migration, invasion and the expression of matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 14 (MMP-14) after transfection of miR-34a mimics or HNF4α siRNA into SH-SY5Y cells were detected by MTT assay, Transwell assay and Western blot assay, respectively. The target relationship between miR-34a and HNF4α was verified by TargetScan online prediction and dual-luciferase assay. Cell proliferation, migration and invasion of SH-SY5Y cells after overexpression of miR-34a and HNF4α were detected. Results The expression level of miR-34a was decreased (p < .05) while the expression level of HNF4α was increased (p < .05) in paediatric neuroblastoma tissues. Over- expression of Mi-34a or knockdown of HNF4α in SH-SY5Y cells could lead to a decreased of cell proliferation, migration, invasion and the expression of MMP-2 and MMP-14 (p < .05). The results of TargetScan online prediction and dual-luciferase assay indicted that HNF4α was a potential target gene for miR-34a. Overexpression of HNF4α could reverse the inhibition of miR-34a on proliferation, migration and invasion of SH-SY5Y cells. Conclusion The expression of miR-34a was down-regulated in paediatric neuroblastoma tissues, and overexpression of miR-34a could inhibit proliferation, migration and invasion of SH-SY5Y cells by targeting HNF4α.

JMJD6 is a tumorigenic factor and therapeutic target in neuroblastoma

Chromosome 17q21-ter is commonly gained in neuroblastoma, but it is unclear which gene in the region is important for tumorigenesis. The JMJD6 gene at 17q21-ter activates gene transcription. Here we show that JMJD6 forms protein complexes with N-Myc and BRD4, and is important for E2F2, N-Myc and c-Myc transcription. Knocking down JMJD6 reduces neuroblastoma cell proliferation and survival in vitro and tumor progression in mice, and high levels of JMJD6 expression in human neuroblastoma tissues independently predict poor patient prognosis. In addition, JMJD6 gene is associated with transcriptional super-enhancers. Combination therapy with the CDK7/super-enhancer inhibitor THZ1 and the histone deacetylase inhibitor panobinostat synergistically reduces JMJD6, E2F2, N-Myc, c-Myc expression, induces apoptosis in vitro and leads to neuroblastoma tumor regression in mice, which are significantly reversed by forced JMJD6 over-expression. Our findings therefore identify JMJD6 as a neuroblastoma tumorigenesis factor, and the combination therapy as a treatment strategy.

Effects of miR-144 on proliferation, apoptosis and cisplatin resistance by targeting MYCN in pediatric neuroblastoma

Objective: To investigate the effects and mechanisms of miR-144 on proliferation, apoptosis and cisplatin (DDP) resistance of neuroblastoma cells. Methods: Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the mRNA expressions of miR-144 and MYCN in neuroblastoma cell lines, including SH-SY5Y and SK-N-SH, and human umbilical vein endothelial cells HUVEC. The miR-negative control, miR-144 mimics, si-negative control, si-MYCN, miR-144 mimics and pcDNA, miR-144 mimics and pcDNA-MYCN co-transfected SH-SY5Y cells were described as miR-NC, miR-144, si-NC, si-MYCN, miR-144+ pcDNA and miR-144+ pcDNA-MYCN group, respectively. The half maximal inhibitory concentration (IC(50)) and cell proliferation were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. The protein expressions of MYCN, p21, cyclin D1, Bax, Bcl-2 were analyzed by western blot. Cell apoptosis was detected by flow cytometry. The cell fluorescence activity was detected by double luciferase reporter gene assay. Results: Compared with HUVEC cells, the expressions of miR-144 in neuroblastoma cells SH-SY5Y and SK-N-SH significantly decreased, while the mRNA and protein expression of MYCN significantly increased. The IC(50) of DDP was 9.16 μg/ml in SH-SY5Y cells. The absorbance value in 490nm (A(490) value) of miR-144 group was 0.30±0.03, significantly lower than 0.46±0.03 of miR-NC group. The cell apoptotic rate of miR-144 group was 26.94%±2.01%, significantly higher than 9.68%±0.52% of miR-NC group. The IC(50) value of DDP in miR-144 group was 2.95±0.26, significantly lower than 9.23±0.61 of miR-NC group. The expressions of p21, cyclin D1, Bax, Bcl-2 in miR-NC and miR-144 group were 2.67±0.19, 0.41±0.04, 2.12±0.21, 0.18±0.01 and 1.01±0.07, 1.00±0.06, 1.00±0.05, 1.00±0.06, respectively, with statistical significance (all P<0.05). Knockdown of MYCN showed the similar effects with those of miR-144 overexpression in SH-SYSY cells. MiR-144 significantly inhibited the fluorescence activity of ectopic MYCN expressing cells and negatively regulated the expression of MYCN. Overexpression of MYCN can reverse the effects of miR-144 on proliferation inhibition, apoptosis promotion and sensitization of SH-SY5Y cells to DDP. Conclusion: MiR-144 inhibits proliferation, promotes apoptosis and enhances the sensitivity of neuroblastoma cells to DDP through targeting MYCN, which provides a potential treatment for neuroblastoma.

Abstract 5209: Efficacious targeting of TERT oncogene rearrangement with BET bromodomain inhibitor and proteasome inhibitor combination therapy

Abstract Background: TERT oncogene rearrangement with transcriptional super-enhancers leads to TERT over-expression in one third of high-risk neuroblastoma patients, and this subtype of neuroblastoma patients shows very poor prognosis. Transcriptional super-enhancers of MYC oncogene are controlled by the BET bromodomain protein BRD4. However, factors important for super-enhancer activity in TERT-rearranged neuroblastoma are unknown. Aims: To examine the role of BRD4 in regulating TERT over-expression and cell proliferation in TERT-rearranged neuroblastoma cells, and to identify the best combination therapy with BRD4 inhibitors and other anticancer agents against TERT-rearranged neuroblastoma. Methods and Results: Knocking down BRD4 with BRD4 siRNAs decreased TERT mRNA and protein expression as well as telomerase activity in TERT-rearranged neuroblastoma cell lines. Alamar blue assays showed that transfection with TERT siRNAs or BRD4 siRNAs considerably reduced cell proliferation. After screening of the Food and Drug Administration-approved oncology drug library, we identified the proteasome inhibitor, carfilzomib, as the compound exerting the best synergistic anticancer effects, in combination with the BRD4 inhibitor OTX-015, against TERT-rearranged neuroblastoma cells. Immunoblot and telomerase activity assays showed that OTX-015 and carfilzomib synergistically decreased TERT protein expression and telomerase activity. Beta-galactosidase and Annexin-V staining of TERT-rearranged neuroblastoma cells revealed that OTX-015 and carfilzomib did not induce senescence but synergistically caused apoptosis, and that transfection with a TERT-over-expression construct rescued the tumour cells from the combination treatment. Importantly, in mice xenografted with TERT-rearranged neuroblastoma cells, combination therapy considerably suppressed tumour progression. Immunoblot analysis of mouse tumour tissues revealed that OTX-015 and carfilzomib dramatically reduced TERT protein expression. Immunohistochemistry analysis showed that OTX-015 and carfilzomib co-operatively reduced the proportion of neuroblastoma cells positively stained with PCNA, indicating that the combination treatment synergistically repressed tumour proliferation. Conclusions: The BET bromodomain protein BRD4 is essential for TERT oncogene expression and cell proliferation in TERT-rearranged neuroblastoma cells. Combination therapy with OTX-015 and carfilzomib considerably reduces TERT expression, induces TERT-rearranged neuroblastoma cell death and tumour growth inhibition, and is a very promising novel strategy for the therapy of TERT-rearranged neuroblastoma. Citation Format: Jingwei Chen, Christopher Nelson, Peiyan Liu, Andrew E. Tee, Bernard Atmadibrata, Karen MacKenzie, Jamie Fletcher, Toby Trahair, Patsie Polly, Roger Reddel, Hilda Pickett, Tao Liu. Efficacious targeting of TERT oncogene rearrangement with BET bromodomain inhibitor and proteasome inhibitor combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5209.