Abstract 5237: The long intergenic noncoding RNA LINC00340 is a neuroblastoma susceptibility gene

Abstract

Abstract Background: Neuroblastoma is a clinically heterogeneous childhood tumor that presents as high-risk disease in 40% of diagnoses, and is fatal in roughly half of these patients despite aggressive multi-modal therapy. In an effort to understand the etiology of high-risk neuroblastoma, our lab previously undertook a genome-wide association study (2101 cases, 4202 controls), which identified a cluster of single-nucleotide polymorphisms on chromosome 6p that correlated with a significantly increased chance of developing of high-risk disease (p < 1e-15). Here we present evidence that these polymorphisms fall within a long intergenic non-coding RNA (LINC00340) termed cancer-associated susceptibility 15 gene (CASC15), which we hypothesize plays a critical role in the development of high-risk neuroblastoma through the dysregulation of neuronal growth and differentiation pathways. Methods: CASC15 expression from patient samples (n=251) was obtained via Affymetrix Human Exon 1.0ST arrays, whereas expression in cell lines was assessed by qRT-PCR. CASC15 isoform sequences were validated through the use of 5′ and 3′ RACE, and subcellular localization was visualized using RNA-FISH. CASC15 depletion was carried out using both siRNA and shRNA constructs, and cell viability and growth kinetics were measured using the CellTiter-Glo and Xcelligence platforms. Gene expression analyses of neuroblastoma cell lines stably silenced for CASC15 was done through hybridization to Human Transcriptome 2.0 arrays followed by gene set enrichment and Ingenuity pathway analysis. Results: Our data demonstrates that patients with high-risk neuroblastoma express significantly less CASC15 than do those with low-risk disease (p < 0.0001), and that CASC15 expression inversely correlates with overall survival (bonf p = 3.1e-05). Expression of CASC15 is variable amongst neuroblastoma cell lines (n= 21), and is predominantly nuclear. Depletion of CASC15 increases the rate of substrate-dependent adherent cellular growth, but has no effect on overall cell number or viability. In contrast, ectopic overexpression of CASC15 in neuroblastoma cell lines is not tolerated. Subsequent observation of cells with stable CASC15 depletion shows overt morphological changes suggestive of a less neuronal phenotype. Gene set expression and pathway analyses confirm that these cells undergo a significant downregulation of neuronal markers, while upregulating cell adhesion molecules. Conclusions: These data suggest that CASC15 may be integral for proper neuronal development, and that loss of CASC15 may leads to a less differentiated cell type, thereby contributing to the tumorigenesis of high-risk neuroblastoma. Citation Format: Mike R. Russell, Annalise Penikis, Derek Oldridge, Maura Diamond, Sharon Diskin, John Maris, Kristina Cole. The long intergenic noncoding RNA LINC00340 is a neuroblastoma susceptibility gene. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5237. doi:10.1158/1538-7445.AM2014-5237