ABSTRACT
The molecular mechanisms underlying the aggressive behavior of MYCN driven neuroblastoma (NBL) is under intense investigation; however, little is known about the impact of this family of transcription factors on the splicing program. Here we used high-throughput RNA sequencing to systematically study the expression of RNA isoforms in stage 4 MYCN-amplified NBL, an aggressive subtype of metastatic NBL. We show that MYCN-amplified NBL tumors display a distinct gene splicing pattern affecting multiple cancer hallmark functions. Six splicing factors displayed unique differential expression patterns in MYCN-amplified tumors and cell lines, and the binding motifs for some of these splicing factors are significantly enriched in differentially-spliced genes. Direct binding of MYCN to promoter regions of the splicing factors PTBP1 and HNRNPA1 detected by ChIP-seq demonstrates that MYCN controls the splicing pattern by direct regulation of the expression of these key splicing factors. Furthermore, high expression of PTBP1 and HNRNPA1 was significantly associated with poor overall survival of stage4 NBL patients (p ≤ 0.05). Knocking down PTBP1, HNRNPA1 and their downstream target PKM2, an isoform of pro-tumor-growth, result in repressed growth of NBL cells. Therefore, our study reveals a novel role of MYCN in controlling global splicing program through regulation of splicing factors in addition to its well-known role in the transcription program. These findings suggest a therapeutically potential to target the key splicing factors or gene isoforms in high-risk NBL with MYCN-amplification.
Subject(s)
Alternative Splicing , Biomarkers, Tumor/genetics , Neuroblastoma/genetics , Nuclear Proteins/genetics , Oncogene Proteins/genetics , RNA, Neoplasm/genetics , Binding Sites , Biomarkers, Tumor/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line, Tumor , Cell Proliferation , Chromatin Immunoprecipitation , Gene Amplification , Gene Expression Regulation, Neoplastic , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , High-Throughput Nucleotide Sequencing , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , N-Myc Proto-Oncogene Protein , Neoplasm Staging , Neuroblastoma/metabolism , Neuroblastoma/mortality , Nuclear Proteins/metabolism , Oncogene Proteins/metabolism , Polypyrimidine Tract-Binding Protein/genetics , Polypyrimidine Tract-Binding Protein/metabolism , Promoter Regions, Genetic , RNA Interference , RNA, Neoplasm/metabolism , Risk Factors , Thyroid Hormones/genetics , Thyroid Hormones/metabolism , Transcription, Genetic , Transfection , Thyroid Hormone-Binding ProteinsABSTRACT
We report the most common single-nucleotide substitution/deletion mutations in favorable histology Wilms tumors (FHWTs) to occur within SIX1/2 (7% of 534 tumors) and microRNA processing genes (miRNAPGs) DGCR8 and DROSHA (15% of 534 tumors). Comprehensive analysis of 77 FHWTs indicates that tumors with SIX1/2 and/or miRNAPG mutations show a pre-induction metanephric mesenchyme gene expression pattern and are significantly associated with both perilobar nephrogenic rests and 11p15 imprinting aberrations. Significantly decreased expression of mature Let-7a and the miR-200 family (responsible for mesenchymal-to-epithelial transition) in miRNAPG mutant tumors is associated with an undifferentiated blastemal histology. The combination of SIX and miRNAPG mutations in the same tumor is associated with evidence of RAS activation and a higher rate of relapse and death.
