<div>Abstract<p>Although neuroblastoma is characterized by numerous recurrent, large-scale chromosomal imbalances, the genes targeted by such imbalances have remained elusive. We have applied whole-genome oligonucleotide array comparative genomic hybridization (median probe spacing 6 kb) to 56 neuroblastoma tumors and cell lines to identify genes involved with disease pathogenesis. This set of tumors was selected for having either 11q loss or <i>MYCN</i> amplification, abnormalities that define the two most common genetic subtypes of metastatic neuroblastoma. Our analyses have permitted us to map large-scale chromosomal imbalances and high-level amplifications at exon-level resolution and to identify novel microdeletions and duplications. Chromosomal breakpoints (<i>n</i> = 467) generating imbalances >2 Mb were mapped to intervals ranging between 6 and 50 kb in size, providing substantial information on each abnormality. For example, breakpoints leading to large-scale hemizygous loss of chromosome 11q were highly clustered and preferentially associated with segmental duplications. High-level amplifications of <i>MYCN</i> were extremely complex, often resulting in a series of discontinuous regions of amplification. Imbalances (<i>n</i> = 540) <2 Mb long were also detected. Although the majority (78%) of these imbalances mapped to segmentally duplicated regions and primarily reflect constitutional copy number polymorphisms, many subtle imbalances were detected that are likely somatically acquired alterations and include genes involved with tumorigenesis, apoptosis, or neural cell differentiation. The most frequent microdeletion involved the <i>PTPRD</i> locus, indicating a possible tumor suppressor function for this gene. (Cancer Res 2006; 66(7): 3673-80)</p></div>
Abstract MicroRNAs are post-transcriptional gene regulators that play a ubiquitous role in cellular functioning. Aberrant expression of miRNAs has been correlated with many cancer types, including neuroblastoma. Neuroblastoma is an often fatal pediatric cancer derived from precursor cells of the sympathetic nervous system. The single most important indicator of unfavourable metastatic disease is amplification of the MYCN oncogenic transcription factor. MiR-335 is significantly down-regulated in MYCN amplified tumors (Bray et al Plos One 2009; 4:e7850) suggesting a possible tumor suppressive function. This study aims to determine if miR-335 functions as a tumor suppressor in neuroblastoma and to elucidate the mechanism by which it elicits this effect. Ectopic up-regulation of miR-335 significantly reduced the motile and invasive capacity of neuroblastoma cell lines. Inversely, down-regulation of miR-335 significantly increased their migratory and invasive potential. mRNA expression profiling arrays in conjunction with the miRNA target prediction algorithm TargetScan identified potential targets of miR-335. Three of these candidate genes, ROCK1, MAPK1 and LRG1, were selected for further investigation because of their known or predicted involvement in cell migration and invasion. Down-regulation of these genes by miR-335 was validated at mRNA and protein levels and direct targeting by miR-335 was confirmed by luciferase reporter assays. siRNA-mediated inhibition of each of these three genes recapitulated the diminished cell migration and invasion associated with miR-335 up-regulation. ROCK1 and MAPK1 are members of two of the major branches of the oncogenic non-canonical TGF-beta signalling pathway, whose signals converge to tightly regulate the phosphorylation or activation status of the motor protein myosin light chain (MLC). LRG1 is a putative TGF-beta pathway member that has been proposed to bind to TGF-beta receptor II. We demonstrate that siRNA mediated inhibition of ROCK1, MAPK1 and LRG1 leads to a significant reduction in phosphorylated levels of MLC protein. Furthermore, we demonstrate that up-regulation of miR-335 inhibits phosphorylation or activation of MLC through targeting of these three genes. Finally, we demonstrate using a MYCN repressible cell line and MYCN ChIP-chip experiments that miR-335 is directly repressed by the oncogenic transcription factor MYCN. Therefore, we conclude that miR-335 is directly repressed by MYCN in MYCN amplified neuroblastoma tumors leading to the increased expression of the oncogenic non-canonical TGF-beta pathway members ROCK1, MAPK1 and LRG1 which function to enhance the metastatic propensity of the tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2290. doi:1538-7445.AM2012-2290
Background MicroRNAs are small molecules which regulate gene expression post-transcriptionally and aberrant expression of several miRNAs is associated with neuroblastoma, a childhood cancer arising from precursor cells of the sympathetic nervous system. Amplification of the MYCN transcription factor characterizes the most clinically aggressive subtype of this disease, and although alteration of p53 signaling is not commonly found in primary tumors, deregulation of proteins involved in this pathway frequently arise in recurrent disease after pharmacological treatment. TH-MYCN is a well-characterized transgenic model of MYCN-driven neuroblastoma which recapitulates many clinicopathologic features of the human disease. Here, we evaluate the dysregulation of miRNAs in tumors from TH-MYCN mice that are either wild-type (TH-MYCN) or deficient (TH-MYCN/p53ERTAM) for the p53 tumor suppressor gene. Principal Findings We analyzed the expression of 591 miRNAs in control (adrenal) and neuroblastoma tumor tissues derived from either TH-MYCN or TH-MYCN/p53ERTAM mice, respectively wild-type or deficient in p53. Comparing miRNA expression in tumor and control samples, we identified 159 differentially expressed miRNAs. Using data previously obtained from human neuroblastoma samples, we performed a comparison of miRNA expression between murine and human tumors to assess the concordance between murine and human expression data. Notably, the miR-17-5p-92 oncogenic polycistronic cluster, which is over-expressed in human MYCN amplified tumors, was over-expressed in mouse tumors. Moreover, analyzing miRNAs expression in a mouse model (TH-MYCN/p53ERTAM) possessing a transgenic p53 allele that drives the expression of an inactive protein, we identified miR-125b-3p and miR-676 as directly or indirectly regulated by the level of functional p53. Significance Our study represents the first miRNA profiling of an important mouse model of neuroblastoma. Similarities and differences in miRNAs expression between human and murine neuroblastoma were identified, providing important insight into the efficacy of this mouse model for assessing miRNA involvement in neuroblastoma and their potential effectiveness as therapeutic targets.
Abstract Neuroblastoma is the most common extra‐cranial solid tumour in children. Natural killer ( NK ) cells are innate lymphocytes that are known to mediate the direct cytotoxicity of neuroblastoma tumour cells. Natural variation in the highly polymorphic killer immunoglobulin‐like receptors ( KIR ) and their cognate human leukocyte antigen ( HLA ) class I ligands results in considerable diversity in NK cell function. As the early onset of neuroblastoma suggests the contribution of genetic factors, we investigated if individual KIR genes, combined KIR gene haplotypes or compound KIR‐HLA ligand genotypes could influence susceptibility to neuroblastoma. Genotype analysis of the KIR genes as well as their three major HLA class I ligand groups, HLA‐C1 , HLA‐C2 and HLA ‐Bw4, was carried out in a cohort of 201 neuroblastoma patients compared with 240 healthy control subjects using polymerase chain reaction with sequence‐specific primers. We found a significant increase in the frequency of KIR2DL2 ( P = 0.019) as well as KIR2DS2 ( P = 0.008) in patients with neuroblastoma compared with the healthy control group. While the incidence of the least inhibitory compound KIR‐HLA ‐C genotype, KIR2DL3 in the presence of HLA‐C1 was slightly reduced in neuroblastoma patients, this did not reach statistical significance ( P = 0.069). In summary, while KIR‐HLA compound genotypes have previously been implicated in predicting treatment outcomes in neuroblastoma, here we show that the presence of the individual KIR genes, KIR2DL2 and KIR2DS2 , irrespective of HLA ‐C genotype is associated with the onset of this embryonal malignancy.
Repetitive DNA sequences have been implicated in the origin of several disease phenotypes, including fragile X syndrome, myotonic dystrophy, and spinal bulbar atrophy. In addition, complex family of chromosome 16-specific low-abundance repetitive (CH16LAR) DNA sequences have been mapped by fluorescence in situ hybridization to regions of chromosome a 16 that undergo breakage/rearrangement in acute nonlymphocytic leukemia (ANLL) cells. It has been hypothesized that these repetitive sequences are causally related to the chromosome rearrangements found in ANLL. Here, we further refine the mapping of CHI 6LAR sequences with respect to the ANLL inversion breakpoints, using a panel of somatic cell hybrids containing 51 different chromosome 16 breakpoints. These studies indicate that CH16LAR sequences at 16p13 are in close proximity to the ANLL short-arm breakpoint region. However, the region containing the highest density of CH16LAR sequences on the long arm appears to be distal to the region where the ANLL long-arm breakpoint has been mapped. These studies further show that CHI 6LAR sequences map in close proximity to FRA16D and FRA16A.
We describe here the derivation, characterization, and use of clonal cadmium-resistant (Cdr) strains of the Chinese hamster cell line CHO which differ in their metallothionein (MT) induction capacity. By nondenaturing polyacrylamide gel electrophoresis, we showed that the stable Cdr phenotype is correlated with the augmented expression of both isometallothioneins (MTI and MTII). In cells resistant to concentrations of CdCl2 exceeding 20 microM, coordinate amplification of genes encoding both isometallothioneins was demonstrated by using cDNA MT-coding sequence probes and probes specific for 3'-noncoding regions of Chinese hamster MTI and MTII genes. Molecular and in situ hybridization analyses supported close linkage of Chinese hamster MTI and MTII genes, which we have mapped previously to Chinese hamster chromosome 3. This suggests the existence of a functionally related MT gene cluster in this species. Amplified Cdr variants expressing abundant MT and their corresponding Cds parental CHO cells should be useful for future studies directed toward elucidating the mechanisms that regulate expression of the isometallothioneins.
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