Project description:High levels of LMO1 expression synergizes with MYCN to accelerate neuroblastomagenesis, enhance disease penetrance and promote widespread metastasis in zebrafish. Transcriptomic analysis of human neuroblasotma cells with programed expression of LMO1 vs vector control or neuroblastoma cells with differential endogenous LMO1 expression revealed that gene signitures affecting tumor cell-extracellular matrix interaction are significantly associated with high levels of LMO1 expression. Our findings provide compelling evidence for a major pathogenic role of LMO1 in MYCN-driven neuroblastoma.
Project description:Neuroblastoma is an embryonal tumor of the peripheral sympathetic nervous system. Elevated expression of the transcription factor LMO1 and the polymorphisms within this gene are associated with the susceptibility to develop neuroblastoma. LMO1 has been implicated as an oncogene in T-cell acute lymphoblastic leukemia; however, the transcriptional targets regulated by this protein in neuroblastoma cells are unknown. Here, we identify the genes and molecular pathways controlled by LMO1 in neuroblastoma cells. ChIP-seq analysis revealed that LMO1-bound regions are frequently co-occupied by GATA3 and MYCN proteins and are associated with active histone marks in neuroblastoma cells. RNA-seq analysis demonstrated that LMO1 regulates gene expression in a tumor type-specific manner. One high-confidence target gene directly regulated by LMO1 and MYCN is ASCL1, which is more highly expressed in adrenergic subtype of neuroblastoma cells as compared to normal neuronal cells. High levels of ASCL1 expression are associated with inferior overall survival in primary human neuroblastoma cases. ChIP-seq analysis identified a regulatory element controlling ASCL1 expression that is bound by LMO1, MYCN and the members of the core regulatory circuitry in neuroblastoma cells. Furthermore, ASCL1 is required for neuroblastoma cell growth and regulates genes responsible for repression of neuronal cell differentiation. Taken together, our results implicate ASCL1 as a critical downstream target of LMO1 in the molecular pathogenesis of neuroblastoma.
Project description:To dissect molecular pathways regulated by LMO1 in neuroblastoma, we performed microarray gene expression profiling in a neuroblastoma cells (SHSY-5Y) after LMO1 knockdown.
Project description:The MYCN locus is amplified in about half of high-risk neuroblastoma tumors. To identify genomic loci occupied by MYCN protein in the MYCN-amplified neuroblastoma cell lines NGP, Kelly and NB-1643, we performed chromatin immunoprecipitation coupled with Next-Generation Sequencing (ChIP-seq) using an anti-MYCN antibody.
Project description:Inducible MYCN-knockdown, followed by RNA-seq analysis in the MYCN-amplified neuroblastoma cell line IMR5-75, reveals profound time-dependent transcriptome changes. For modulation of MYCN levels, stable neuroblastoma cell models were used where MYCN can be downregulated via vector-based hairpin RNA induction upon addition of 1µg/ml tetracycline (IMR5-75-shMYCN. From cells treated either with tetracycline or solvent (ethanol), RNA was isolated at time points 6 hours, 12 hours and 24 hours. Experiments were done in duplicates. RNA was sequenced.
Project description:Inducible MYCN-knockdown, followed by RNA-seq analysis in the MYCN-amplified neuroblastoma cell line IMR5-75, reveals profound time-dependent transcriptome changes.
Project description:Purpose: Identify new targets in MYCN-amplified Neuroblastoma Methods: ChIP-Seq experiments were performed on Kelly and LAN-1 neuroblastoma cells by using the following antibodies: anti-EZH2 (Cell Signaling 5246S); anti-H3K27me3 (Millipore 07-449); anti-H3K4me3 (Abcam ab8580). We evaluated the global EZH2 PRC2-dependence by identifiying direct genome-wide target genes for EZH2, H3K27me3 and H3K4me3. Results: We found that EZH2 serves a PRC2-dependent function in neuroblastoma, repressing neuronal differentiation. Moreover, EZH2-regulated genes were strongly repressed in MYCN-amplified and high-risk primary tumors. Conclusion: Our study supports testing EZH2 inhibitors in patients with MYCN-amplified neuroblastoma.
Project description:RNA-seq upon TBX2, MYCN or combination of TBX2 and MYCN knockdown in the neuroblastoma cell line IMR-5/75. Cells were transduced with two different shRNAs (shTBX2_2 and shTBX2_4) targeting TBX2 and a non-targeting control (NTC), and selected with puromycin. Cells were treated with doxycycline for shMYCN induction (with DOX or not). Analysis was performed three days upon TBX2 knockdown and two days upon MYCN knockdown, including six biological replicates per condition.
