Hsa-miR-323a-3p functions as a tumor suppressor and targets STAT3 in neuroblastoma cells.

Background: Studies conducted in the last decades have revealed a role for the non-coding microRNAs (miRNAs) in cancer development and progression. Several miRNAs within the chromosome region 14q32, a region commonly deleted in cancers, are associated with poor clinical outcome in the childhood cancer neuroblastoma. We have previously identified miR-323a-3p from this region to be downregulated in chemotherapy treated neuroblastoma cells compared to pre-treatment cells from the same patients. Furthermore, in neuroblastoma tumors, this miRNA is downregulated in advanced stage 4 disease compared to stage 1-2. In this study, we attempt to delineate the unknown functional roles of miR-323a-3p in neuroblastoma. Methods: Synthetic miRNA mimics were used to overexpress miR-323a-3p in neuroblastoma cell lines. To investigate the functional roles of miR-323a-3p, cell viability assay, flow cytometry, reverse transcription-quantitative polymerase chain reaction, luciferase reporter assay and western blot were conducted on the neuroblastoma cell lines Kelly, SH-SY5Y and SK-N-BE(2)-C. Results: Ectopic expression of miR-323a-3p resulted in marked reduction of cell viability in Kelly, SH-SY5Y and SK-N-BE(2)-C by causing G1-cell cycle arrest in Kelly and SH-SY5Y and apoptosis in all the cell lines tested. Furthermore, mRNA and protein levels of signal transducer and activator of transcription 3 (STAT3) were reduced upon miR-323a-3p overexpression. A direct binding of the miR-323a-3p to the 3'UTR of STAT3 was experimentally validated by luciferase reporter assay, where miR-323a-3p reduced luminescent signal from full length STAT3 3'UTR luciferase reporter, but not from a reporter with mutation in the predicted seed sequence. Conclusions: miR-323a-3p inhibits growth of neuroblastoma cell lines through G1-cell cycle arrest and apoptosis, and the well-known oncogene STAT3 is a direct target of this miRNA.

Clinically Relevant Biomarker Discovery in High-Risk Recurrent Neuroblastoma.

Neuroblastoma is a pediatric cancer of the developing sympathetic nervous system. High-risk neuroblastoma patients typically undergo an initial remission in response to treatment, followed by recurrence of aggressive tumors that have become refractory to further treatment. The need for biomarkers that can select patients not responding well to therapy in an early phase is therefore needed. In this study, we used next generation sequencing technology to determine the expression profiles in high-risk neuroblastoma cell lines established before and after therapy. Using partial least squares-discriminant analysis (PLS-DA) with least absolute shrinkage and selection operator (LASSO) and leave-one-out cross-validation, we identified a panel of 55 messenger RNAs and 17 long non-coding RNAs (lncRNAs) which were significantly altered in the expression between cell lines isolated from primary and recurrent tumors. From a neuroblastoma patient cohort, we found 20 of the 55 protein-coding genes to be differentially expressed in patients with unfavorable compared with favorable outcome. We further found a twofold increase or decrease in hazard ratios in these genes when comparing patients with unfavorable and favorable outcome. Gene set enrichment analysis (GSEA) revealed that these genes were involved in proliferation, differentiation and regulated by Polycomb group (PcG) proteins. Of the 17 lncRNAs, 3 upregulated (NEAT1, SH3BP5-AS1, NORAD) and 3 downregulated lncRNAs (DUBR, MEG3, DHRS4-AS1) were also found to be differentially expressed in favorable compared with unfavorable outcome. Moreover, using expression profiles on both miRNAs and mRNAs in the same cohort of cell lines, we found 13 downregulated and 18 upregulated experimentally observed miRNA target genes targeted by miR-21, -424 and -30e, -29b, -138, -494, -181a, -34a, -29b, respectively. The advantage of analyzing biomarkers in a clinically relevant neuroblastoma model system enables further studies on the effect of individual genes upon gene perturbation. In summary, this study identified several genes, which may aid in the prediction of response to therapy and tumor recurrence.

Inhibitors of ribosome biogenesis repress the growth of MYCN-amplified neuroblastoma.

Abnormal increases in nucleolar size and number caused by dysregulation of ribosome biogenesis has emerged as a hallmark in the majority of spontaneous cancers. The observed ribosome hyperactivity can be directly induced by the MYC transcription factors controlling the expression of RNA and protein components of the ribosome. Neuroblastoma, a highly malignant childhood tumor of the sympathetic nervous system, is frequently characterized by MYCN gene amplification and high expression of MYCN and c-MYC signature genes. Here, we show a strong correlation between high-risk disease, MYCN expression, poor survival, and ribosome biogenesis in neuroblastoma patients. Treatment of neuroblastoma cells with quarfloxin or CX-5461, two small molecule inhibitors of RNA polymerase I, suppressed MycN expression, induced DNA damage, and activated p53 followed by cell cycle arrest or apoptosis. CX-5461 repressed the growth of established MYCN-amplified neuroblastoma xenograft tumors in nude mice. These findings suggest that inhibition of ribosome biogenesis represent new therapeutic opportunities for children with high-risk neuroblastomas expressing high levels of Myc.

Hsa-miR-376c-3p targets Cyclin D1 and induces G1-cell cycle arrest in neuroblastoma cells.

High-risk neuroblastoma is the most aggressive form of cancer in children. The estimated survival of children with high-risk neuroblastoma is 40-50% compared with low and intermediate risk neuroblastoma, which is >98 and 90-95%, respectively. In addition, patients with high-risk neuroblastoma often experience relapse following intensive treatments with standard chemotherapeutic drugs. Therefore alternative strategies are required to address this problem. MicroRNAs (miRNAs/miRs) are small, endogenously expressed non-coding RNAs, which when deregulated have been demonstrated to serve significant roles in the tumorigenesis of a number of different types of cancer. Results from a previous deep sequencing study identified 22 downregulated miRNAs from the 14q32 miRNA cluster differentially expressed in neuroblastoma cell lines isolated from 6 patients at diagnosis and at relapse following intensive treatments. miR-376c-3p is one of the 22 miRNAs that was downregulated in the majority of the cell lines isolated from patients post treatment. The present study employed reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to quantify the basic expression of miR-376c-3p in 6 neuroblastoma cell lines. The functional role of miR-376c-3p in the neuroblastoma cell lines was evaluated by alamar blue-cell viability and propidium iodide-flow cytometric assays. In addition, luciferase reporter assays, RT-qPCR and western blotting were performed to identify and quantify the targets of miR-376c-3p in neuroblastoma cell lines. Ectopic expression of miR-376c-3p led to significant inhibition of cell viability and G1-cell cycle arrest in multiple neuroblastoma cell lines by reducing the expression of cyclin D1, an oncogene critical for neuroblastoma pathogenesis. The results of the present study provide novel insights into the functional role of miR-376c-3p and suggest new approaches for the treatment of neuroblastoma.

MicroRNA-193b-3p represses neuroblastoma cell growth via downregulation of Cyclin D1, MCL-1 and MYCN.

Neuroblastoma is the most common diagnosed tumor in infants and the second most common extracranial tumor of childhood. The survival rate of patients with high-risk neuroblastoma is still very low despite intensive multimodal treatments. Therefore, new treatment strategies are needed. In recent years, miRNA-based anticancer therapy has received growing attention. Advances in this novel treatment strategy strongly depends on the identification of candidate miRNAs with broad-spectrum antitumor activity. Here, we identify miR-193b as a miRNA with tumor suppressive properties. We show that miR-193b is expressed at low levels in neuroblastoma cell lines and primary tumor samples. Introduction of miR-193b mimics into nine neuroblastoma cell lines with distinct genetic characteristics significantly reduces cell growth in vitro independent of risk factors such as p53 functionality or MYCN amplification. Functionally, miR-193b induces a G1 cell cycle arrest and cell death in neuroblastoma cell lines by reducing the expression of MYCN, Cyclin D1 and MCL-1, three important oncogenes in neuroblastoma of which inhibition has shown promising results in preclinical testing. Therefore, we suggest that miR-193b may represent a new candidate for miRNA-based anticancer therapy in neuroblastoma.

Next generation sequencing of microRNAs from isogenic neuroblastoma cell lines isolated before and after treatment.

Neuroblastoma is a pediatric cancer of the developing sympathetic nervous system. High risk neuroblastoma patients typically undergo an initial remission in response to treatment, followed by recurrence of aggressive tumors that have become refractory to further treatment. Recent works have underlined the involvement of microRNAs (miRNAs) in neuroblastoma development and evolution of drug resistance. In this study we have used deep sequencing technology to identify miRNAs differentially expressed in neuroblastoma cell lines isolated from 6 patients at diagnosis and at relapse after intensive treatments. This approach revealed a panel of 42 differentially expressed miRNAs, 8 of which were upregulated and 34 were downregulated. Most strikingly, the 14q32 miRNA clusters encode 22 of the downregulated miRNAs. Reduced expression of 14q32 miRNAs in tumors associated with poor prognosis factors was confirmed in a cohort consisting of 226 primary neuroblastomas. In order to gain insight into the nature of the genes that may be affected by the differentially expressed miRNAs we utilized Ingenuity Pathway Analysis (IPA). This analysis revealed several biological functions and canonical pathways associated with cancer progression and drug resistance. The results of this study contribute to the identification of miRNAs involved in the complex processes of surviving therapeutic treatment and developing drug resistance in neuroblastoma.

Exosome-like Extracellular Vesicles from MYCN-amplified Neuroblastoma Cells Contain Oncogenic miRNAs.

BACKGROUND: In recent years, evidence has accumulated indicating that both normal and cancer cells communicate via the release and delivery of macromolecules packed into extracellular membrane vesicles. MATERIALS AND METHODS: We isolated nano-sized extracellular vesicles from MYCN-amplified neuroblastoma cell lines using ultracentrifugation and exosome precipitation (Exoquick) protocols. These vesicles were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis and western blotting. Exosomal miRNA profiles were obtained using a reverse transcription-polymerase chain reaction (RT-PCR) ready-to-use panel measuring a total of 742 miRNAs. RESULTS: In this study, we showed that MYCN-amplified neuroblastoma cell lines secrete populations of miRNAs inside small exosome-like vesicular particles. These particles were shown to be taken-up by recipient cells. By profiling the miRNA content, we demonstrated high expression of a group of established oncomirs in exosomes from two MYCN-amplified neuroblastoma cell lines. Despite the fact that other studies have demonstrated the ability of exosomal miRNAs both to repress mRNA targets and to stimulate Toll-like receptor-8 (TLR8) signaling in recipient cells, we did not observe these effects with exosomes from MYCN-amplified neuroblastoma cells. However, functional enrichment analysis reveals that mRNA targets of highly expressed exosomal miRNAs are associated with a range of cellular and molecular functions related to cell growth and cell death. CONCLUSION: MYCN-amplified neuroblastoma cell lines secrete exosome-like particles containing oncogenic miRNAs. This work showed for the first time that neuroblastoma cells secrete exosome-like particles containing miRNAs with potential roles in cancer progression. These findings indicate a new way for MYCN-amplified neuroblastoma cells to interact with the tumor environment.

Conditional expression of retrovirally delivered anti-MYCN shRNA as an in vitro model system to study neuronal differentiation in MYCN-amplified neuroblastoma.

BACKGROUND: Neuroblastoma is a childhood cancer derived from immature cells of the sympathetic nervous system. The disease is clinically heterogeneous, ranging from neuronal differentiated benign ganglioneuromas to aggressive metastatic tumours with poor prognosis. Amplification of the MYCN oncogene is a well established poor prognostic factor found in up to 40% of high risk neuroblastomas.Using neuroblastoma cell lines to study neuronal differentiation in vitro is now well established. Several protocols, including exposure to various agents and growth factors, will differentiate neuroblastoma cell lines into neuron-like cells. These cells are characterized by a neuronal morphology with long extensively branched neurites and expression of several neurospecific markers. RESULTS: In this study we use retrovirally delivered inducible short-hairpin RNA (shRNA) modules to knock down MYCN expression in MYCN-amplified (MNA) neuroblastoma cell lines. By addition of the inducer doxycycline, we show that the Kelly and SK-N-BE(2) neuroblastoma cell lines efficiently differentiate into neuron-like cells with an extensive network of neurites. These cells are further characterized by increased expression of the neuronal differentiation markers NFL and GAP43. In addition, we show that induced expression of retrovirally delivered anti-MYCN shRNA inhibits cell proliferation by increasing the fraction of MNA neuroblastoma cells in the G1 phase of the cell cycle and that the clonogenic growth potential of these cells was also dramatically reduced. CONCLUSION: We have developed an efficient MYCN-knockdown in vitro model system to study neuronal differentiation in MNA neuroblastomas.

Inhibition of gene function in mammalian cells using short-hairpin RNA (shRNA).

RNAi is now the preferred method for silencing gene expression in a variety of systems. In this chapter we describe the procedure for applying short-hairpin RNA (shRNA) to study gene function. Detailed descriptions of target site selection, shRNA construction, shRNA transfection and target knockdown validation are included.

Comparison of RNAi efficiency mediated by tetracycline-responsive H1 and U6 promoter variants in mammalian cell lines.

Conditional expression of short hairpin RNAs (shRNAs) to knock down target genes is a powerful tool to study gene function. The most common inducible expression systems are based on tetracycline-regulated RNA polymerase III promoters. During the last years, several tetracycline-inducible U6 and H1 promoter variants have been reported in different experimental settings showing variable efficiencies. In this study, we compare the most common variants of these promoters in several mammalian cell lines. For all cell lines tested, we find that several inducible U6 and H1 promoters containing single tetracycline operator (tetO) sequences show high-transcriptional background in the non-induced state. Promoter variants containing two tetO sequences show tight suppression of transcription in the non-induced state, and high tet responsiveness and high gene knockdown efficiency upon induction in all cell lines tested. We report a variant of the H1 promoter containing two O2-type tetO sequences flanking the TATA box that shows little transcriptional background in the non-induced state and up to 90% target knockdown when the inducer molecule (dox-doxycycline) is added. This inducible system for RNAi-based gene silencing is a good candidate for use both in basic research on gene function and for potential therapeutic applications.

The antimicrobial peptide, lactoferricin B, is cytotoxic to neuroblastoma cells in vitro and inhibits xenograft growth in vivo.

Antimicrobial peptides have been shown to exert cytotoxic activity towards cancer cells through their ability to interact with negatively charged cell membranes. In this study the cytotoxic effect of the antimicrobial peptide, LfcinB was tested in a panel of human neuroblastoma cell lines. LfcinB displayed a selective cytotoxic activity against both MYCN-amplified and non-MYCN-amplified cell lines. Non-transformed fibroblasts were not substantially affected by LfcinB. Treatment of neuroblastoma cells with LfcinB induced rapid destabilization of the cytoplasmic membrane and formation of membrane blebs. Depolarization of the mitochondria membranes and irreversible changes in the mitochondria morphology was also evident. Immuno- and fluorescence-labeled LfcinB revealed that the peptide co-localized with mitochondria. Furthermore, treatment of neuroblastoma cells with LfcinB induced cleavage of caspase-6, -7 and -9 followed by cell death. However, neither addition of the pan-caspase inhibitor, zVAD-fmk, or specific caspase inhibitors could reverse the cytotoxic effect induced by LfcinB. Treatment of established SH-SY-5Y neuroblastoma xenografts with repeated injections of LfcinB resulted in significant tumor growth inhibition. These results revealed a selective destabilizing effect of LfcinB on two important targets in the neuroblastoma cells, the cytoplasmic- and the mitochondria membrane.

Interferon-γ Sensitises Human Osteosarcoma Cells to Trail-mediated Apoptosis.

BACKGROUND: Nearly half of all patients with osteosarcoma are still not cured by the currently employed multimodal treatment. New strategies are therefore needed to further improve the outcome. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2) is able to induce programmed cell death in transformed cells, while normal cells remain unaffected. MATERIALS AND METHODS: We have investigated the effect of TRAIL in combination with IFN-γ on four human osteosarcoma cell lines; Saos-2, U2OS, KPDXM and OHS, and on one normal human fibroblast cell line, MRC-5. RESULTS: One of the four osteosarcoma cell lines was TRAIL-resistant, but was sensitised to TRAIL-mediated cell death upon pre-incubation with IFN-γ. In two of the three TRAIL-sensitive cell lines, the effect of TRAIL was enhanced by IFN-γ. The normal human fibroblast cell line MRC-5 was not affected by treatment with TRAIL and IFN-γ. A caspase cascade involving the activation of caspase-8, caspase-7 and PARP was associated with the onset of apoptosis in the osteosarcoma cell lines. Apoptosis was partly inhibited by the addition of caspase inhibitors zVADfmk (general inhibitor) and zIETDfmk (selective caspase-8 inhibitor). These findings further emphasize the important role of the caspases in cell death signalling. CONCLUSION: Our results show that treatment with both IFN-γ and TRAIL efficiently induces cell death in osteosarcoma cell lines and should be further investigated as a potential future therapy.