20 research outputs found

    Establishment of a new MYCN-driven murine neuroblastoma model and analysis of tumorsuppressive mircoRNAs

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    Das Neuroblastom ist der h√§ufigste solide Tumor des Kindesalters und verantwortlich f√ľr ca. 15% aller p√§diatrisch-onkologischen Todesf√§lle. Eine Amplifikation des Onkogens MYCN, durch welche die prognostisch ung√ľnstigste Patientengruppe definiert ist, liegt in 20% aller Neuroblastome vor. Im Rahmen dieser Arbeit wurde ein neues Cre-konditionales MYCN-vermitteltes Mausmodell (LSL-MYCN) entwickelt. Durch die Kreuzung mit Dbh-iCre M√§usen wurde die Expression des MYCN Transgens auf Dopamin-beta-Hydroxylase exprimierende Zellen beschr√§nkt, was zur Entwicklung von Tumoren der Nebennieren, der oberen Zervikalganglien und des Ganglion coeliacum f√ľhrte. Die Tumore wiesen sowohl die f√ľr das Neuroblastom spezifischen kleinen, blauen und runden Zellen als auch neurosekretorische Vesikel auf. Weiterhin konnte eine signifikante √úberexpression von humanem MYCN und spezifischer Neuroblastommarker detektiert werden. Genomische Aberrationen sowie mRNA und mikroRNA (miRNA) Expressions-Profile entsprachen denen humaner Neuroblastome. MiRNAs sind kurze, nicht-kodierende RNA Molek√ľle, die durch Bindung an mRNAs deren Stabilit√§t und Translation regulieren. Die Bedeutung von miRNAs in der Tumorbiologie ergibt sich aus der Regulation von Onkogenen und Tumorsuppressorgenen. In humanen prim√§ren Neuroblastomen war die niedrige Expression der miRNAs miR-542-3p und miR-137 mit ung√ľnstiger Prognose assoziiert und eine Herunterregulation von miR-542-3p konnte auch in Tumoren des LSL MYCN;Dbh-iCre Modells beobachtet werden. Die Funktion beider miRNAs wurde in vitro in pr√§klinischen Modellsystemen des humanen Neuroblastoms detailliert untersucht. Die ektope Expression der miRNAs reduzierte die Zellviabilit√§t und Proliferation, erh√∂hte die Apoptoserate und induzierte einen Zellzyklusarrest. Weiterhin wurde die direkte Bindung der vorhergesagten Ziel-mRNAs KDM1A (miR-137) und Survivin (miR-542-3p) mittels Luziferase Reporter Analysen validiert und die Regulation sowohl auf mRNA- als auch auf Proteinebene verifiziert. Die Herunterregulation der Zielgene in Neuroblastomzellen stellte eine Ph√§nokopie der Effekte einer exogenen Expression der jeweiligen miRNA dar. Die Spezifit√§t der miRNA-vermittelten Regulation wurde durch ektope Expression des Zielgens mit mutierter miRNA-Bindestelle nachgewiesen. Eine Therapiesimulation in Neuroblastom-Xenografts mit miR-542-3p-beladenen Nanopartikeln zeigte sowohl eine Herunterregulation des Zielproteins Survivin als auch eine Reduktion der Proliferation und Erh√∂hung der Apoptoserate in den Tumorzellen. Die Etablierung des neuen LSL-MYCN;Dbh-iCre Neuroblastom-Mausmodells kann dabei helfen sowohl die Pathogenese des Neuroblastoms besser zu verstehen als auch potentiell Neuroblastom-relevante Gene und miRNAs zu identifizieren. Eine Weiterentwicklung dieses Modells durch Kombination mit anderen Mausmodellen, die potentiell Neuroblastom-relevante Gene regulieren, k√∂nnte im Zusammenspiel mit neuen Behand¬¨lungsstrategien Einblicke in die Wirksamkeit von k√ľnftigen Neuroblastomtherapien geben. Die Behandlung humaner Neuroblastompatienten mit tumorsuppressiven miRNAs, wie miR-542-3p und miR-137, k√∂nnte ein m√∂glicher Ansatz sein den aggressiven Verlauf dieser Erkrankung zu reduzieren.Neuroblastoma is a common solid tumor of infancy and accounts for more than 15% of pediatric cancer deaths. Amplification of MYCN oncogene defines the most aggressive subtype of neuroblastoma with poor prognosis. In the present study, a new Cre-conditional MYCN-driven transgenic mouse model was generated to analyse tumorigenesis in vivo. When targeting MYCN expression to the neural crest in transgenic LSL-MYCN;Dbh-iCre mice, palpable tumors developed, which arose predominantly from both adrenals, but also from superior cervical ganglia and ganglion coeliacum. LSL MYCN;Dbh-iCre-induced tumors consisted of small round blue cells and possessed neurosecretory vesicles. Expression of human MYCN and specific neuroblastoma marker genes was high in tumors. Furthermore, murine neuroblastomas recapitulated genomic aberrations and MYCN mRNA as well as microRNA (miRNA) signatures described for human neuroblastoma. MiRNAs are small noncoding RNA molecules that bind target mRNAs resulting in translational repression or degradation of the mRNA. In cancer, they can act as tumor suppressors or oncogenes. In human primary neuroblastomas, low expression of miR-542-p and miR-137 was highly correlated with unfavourable prognosis and miR-542-3p was upregulated in murine LSL MYCN;Dbh-iCre tumors. Exogenous expression of these miRNAs in established neuroblastoma cell lines reduced viability and proliferation, increased apoptosis and induced cell cycle arrest. Furthermore, direct interaction be¬¨tween the predicted targets survivin (miR-542-3p) and KDM1A (miR-137) was validated by luciferase reporter assays and regulation was demonstrated on mRNA as well as protein level. Ectopic expression of miR-542-3p or miR-137 phenocopied the knock¬¨down of respective targets. In addition, we were able to validate a direct interaction between miRNAs and their targets by performing rescue experiments. Treatment of mice bearing neuroblastoma xenografts with miR-542-3p-loaded nanoparticles resulted in downregulation of survivin and induced reduced tumor cell proliferation as well as increased apoptosis. In conclusion, the novel LSL MYCN;Dbh-iCre neuroblastoma mouse model contribute to a better understanding of MYCN-driven neuroblastoma and to identification of relevant genes and miRNAs. Combination with other transgenic models, regulating potential relevant genes, could provide new insights into neuroblastoma tumorigenesis. Restoring function of tumor suppressive miRNAs, such as miR-542-3p or miR-137, could represent a feasible approach to reduce neuroblastoma aggressiveness

    RITA displays anti-tumor activity in medulloblastomas independent of TP53 status

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    Current therapy of medulloblastoma, the most common malignant brain tumor of childhood, achieves 40-70% survival. Secondary chemotherapy resistance contributes to treatment failure, where TP53 pathway dysfunction plays a key role. MDM2 interaction with TP53 leads to its degradation. Reactivating TP53 functionality using small-molecule inhibitors, such as RITA, to disrupt TP53-MDM2 binding may have therapeutic potential. We show here that RITA decreased viability of all 4 analyzed medulloblastoma cell lines, regardless of TP53 functional status. The decrease in cell viability was accompanied in 3 of the 4 medulloblastoma cell lines by accumulation of TP53 protein in the cells and increased CDKN1A expression. RITA treatment in mouse models inhibited medulloblastoma xenograft tumor growth. These data demonstrate that RITA treatment reduces medulloblastoma cell viability in both in vitro and in vivo models, and acts independently of cellular TP53 status, identifying RITA as a potential therapeutic agent to treat medulloblastoma

    HBP1 downregulation through mutant ALK represents a novel mechanism for cooperative MYCN activation in neuroblastoma

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    Introduction: MYCN signaling plays a key role in initiation and progression of neuroblastoma (NB). Mutant ALK is implicated in accelerated MYCN-driven NB formation in mouse models. Combined MYCN-amplified/mutant ALK NBs have very poor outcome. Finally, recent data showed that ALK mutations frequently occur in relapsed tumors suggesting a specific role for ALK signaling in therapy resistance. Previous studies showed that mutant ALK activates MYCN signaling through transcriptional regulation and protein stabilization. We recently identified a third major HBP1-controlled mechanism of ALK-mediated MYCN activation. Methods: Pathway analysis of HBP1 regulation was done by in vitro analysis of transcriptional response of NB cells to compounds targeting ALK, PI3K/AKT and MAPK signaling. The SHEP cell line with inducible miR-17‚ąľ92 was used to investigate HBP1 regulation. HBP1 was modulated in the NGP cell line in order to study transcriptional networks. Data mining was performed in R using available algorithms. Results: HBP1 is regulated through PI3K/AKT-FOXO3a signaling downstream of mutant ALK and a miR-17‚ąľ92 controlled negative feedback loop, as miR-17‚ąľ92 is positively regulated by MYCN and down-regulates HBP1. EGCG (epigallocatechin gallate) was selected as a tool compound as it is known to up-regulate HBP1 (Kim et al, 2006, JBC). We demonstrate synergistic effects of combined JQ1/EGCG administration in mouse xenografts with significant growth delay in this combination-treated group. Discussion: HBP1 is an important novel NB suppressor integrated into a complex regualtory network governed by ALK and MYCN signaling and offering a novel entry point for MYCN drugging in NB
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