Clinically Relevant Biomarker Discovery in High-Risk Recurrent Neuroblastoma

Source at https://doi.org/10.1177/1176935119832910.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

Endogenous IL-1 receptor antagonist restricts healthy and malignant myeloproliferation

Here we explored the role of interleukin-1β (IL-1β) repressor cytokine, IL-1 receptor antagonist (IL-1rn), in both healthy and abnormal hematopoiesis. Low IL-1RN is frequent in acute myeloid leukemia (AML) patients and represents a prognostic marker of reduced survival. Treatments with IL-1RN and the IL-1β monoclonal antibody canakinumab reduce the expansion of leukemic cells, including CD34+ progenitors, in AML xenografts. In vivo deletion of IL-1rn induces hematopoietic stem cell (HSC) differentiation into the myeloid lineage and hampers B cell development via transcriptional activation of myeloid differentiation pathways dependent on NFκB. Low IL-1rn is present in an experimental model of pre-leukemic myelopoiesis, and IL-1rn deletion promotes myeloproliferation, which relies on the bone marrow hematopoietic and stromal compartments. Conversely, IL-1rn protects against pre-leukemic myelopoiesis. Our data reveal that HSC differentiation is controlled by balanced IL-1β/IL-1rn levels under steady-state, and that loss of repression of IL-1β signaling may underlie pre-leukemic lesion and AML progression

Endogenous IL-1 receptor antagonist restricts healthy and malignant myeloproliferation.

Here we explored the role of interleukin-1β (IL-1β) repressor cytokine, IL-1 receptor antagonist (IL-1rn), in both healthy and abnormal hematopoiesis. Low IL-1RN is frequent in acute myeloid leukemia (AML) patients and represents a prognostic marker of reduced survival. Treatments with IL-1RN and the IL-1β monoclonal antibody canakinumab reduce the expansion of leukemic cells, including CD34+ progenitors, in AML xenografts. In vivo deletion of IL-1rn induces hematopoietic stem cell (HSC) differentiation into the myeloid lineage and hampers B cell development via transcriptional activation of myeloid differentiation pathways dependent on NFκB. Low IL-1rn is present in an experimental model of pre-leukemic myelopoiesis, and IL-1rn deletion promotes myeloproliferation, which relies on the bone marrow hematopoietic and stromal compartments. Conversely, IL-1rn protects against pre-leukemic myelopoiesis. Our data reveal that HSC differentiation is controlled by balanced IL-1β/IL-1rn levels under steady-state, and that loss of repression of IL-1β signaling may underlie pre-leukemic lesion and AML progression.We thank K. Tasken, J. Saarela and the NCMM at the University of Oslo (UiO), S. Kanse (UiO) and B. Smedsrød (UiT), for access to facilities. We acknowledge Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital (Bergen, Norway) and R. Hovland for karyotyping, FISH, translocation and DNA analyses of AML and MDS patients included in this study, and Department of Pathology, Oslo University Hospital (Oslo, Norway) and S. Spetalen for deep sequencing. L.M. Gonzalez, L.T. Eliassen, X. Zhang, M. Ristic and other members of L. Arranz group, O.P. Rekvig, R. Doohan, L.D. Håland, M.I. Olsen, A. Urbanucci, J. Landskron, K.B. Larsen, R.A. Lyså and UiT Advanced Microscopy Core Facility, UiO and UiT Comparative Medicine Units, for assistance. P. Garcia and S. Mendez-Ferrer for providing NRASG12D and Nes-gfp mice, respectively. P. Garcia and L. Kurian for careful reading of the manuscript. E. Tenstad (Science Shaped) for artwork in schematics. We would also like to thank the AML and MDS patients, and healthy volunteers, who donated biological samples. Our work is supported by a joint meeting grant of the Northern Norway Regional Health Authority, the University Hospital of Northern Norway (UNN) and UiT (Strategisk-HN06-14), Young Research Talent grants from the Research Council of Norway, (Stem Cell Program, 247596; FRIPRO Program, 250901), and grants from the Norwegian Cancer Society (6765150), the Northern Norway Regional Health Authority (HNF1338-17), and the Aakre-Stiftelsen Foundation (2016/9050) to L.A. Vav-Cre NRASG12D experiments were supported by NIH grant R01CA152108 to J.Z.S

Non-state actors in world politics : a case study on how transnational advocacy networks seek to influence the Arms Trade Treaty

Theory on international society is often criticized for disregarding the agency and influence of non-state actors. This paper analyzes the ways in which non-governmental organizations seek political influence in the United Nations, based on a case study of the Control Arms campaign in the period 2008-2010. The method is a qualitative, iterative approach, where propositions from recent theory on transnational advocacy networks are matched with data from informants and internal documents to highlight congruent and divergent aspects. It is argued that the findings warrant further research on the various strategies employed by non-state actors in the international arena

mir-34a targets cell cycle genes CCND1 (cyclin D1) and MYCN, while mir-21 does not target PTEN and PDCD4 in neuroblastoma

Neuroblastoma is an embryonal cancer of the post-sympathetic nervous system and is the most frequent extra-cranial solid tumor in childhood. Two important microRNAs, the tumor suppressor mir-34a and the oncogenic mir-21, have been found to increase during MYCN-knockdown induced differentiation. However their distinct roles are not clear. This thesis employs a method to identify the mRNA targets of mir-21 and mir-34a by the capture of biotin-labeled microRNA-mRNA complexes. The function of the biotin-labeled microRNA duplexes mir-21 and mir-34a were proven to be biologically active by targeting of the antisense mir-21 and MYCN 3’ UTR cloned into the firefly luciferase, respectively. Further, these luciferase vectors were used to prove the applicability of the pull-down approach where mir-21 and mir-34a enriched for their respective targets. A biotinylated, negative control did not enrich for the luciferase transcript nor did the biotinylated mimics enrich for HPRT1 (a housekeeping gene). Moreover, the method was used to search for endogenous targets in the MYCN-amplified neuroblastoma cell line SK-N-BE(2)c. The pull-down results of mir-34a yielded high enrichment of MYCN and CCND1 (cyclin D1). Additionally, SIRT1 was a probable target of mir-34a. Two mRNAs that previously were shown to be targeted by mir-21 in human cancers, PTEN and PDCD4, were not enriched from neuroblastoma cell extracts. Previous results from our lab have also shown that pre-mir-21 over-expression do not affect PTEN and PDCD4 protein levels. Over-expression of the biotinylated mir-21 duplex did neither affect mRNA levels of PTEN nor PDCD4. mir-21 was as such concluded to not target PTEN and PDCD4 in the human neuroblastoma cell line SK-N-BE(2)c. Moreover, mir-34a over-expression in BE(2)c showed a 4-fold reduction of MYCN and a 20 % increase in CCND1. Mechanisms of mir-34a were therefore believed to repress and perhaps direct CCND1 to P-bodies. mir-34a over-expression resulted in the respective 1.75 and 7.8 fold increase of the two neuronal differentiation markers NPY and GAP43. This was conclusive with the tumor suppressing role of mir-34a. Additionally, the role of mir-34a in neuroblastoma is discussed in detail. Concluding, this thesis demonstrates how biotinylated microRNA duplexes can be used to identify miRNA-mRNA targets

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