CASC15-S Is a Tumor Suppressor lncRNA at the 6p22 Neuroblastoma Susceptibility Locus

Abstract

Chromosome 6p22 was identified recently as a neuroblastoma susceptibility locus, but its mechanistic contributions to tumorigenesis are as yet undefined. Here we report that the most highly significant single-nucleotide polymorphism (SNP) associations reside within CASC15, a long noncoding RNA that we define as a tumor suppressor at 6p22. Low-level expression of a short CASC15 isoform (CASC15-S) associated highly with advanced neuroblastoma and poor patient survival. In human neuroblastoma cells, attenuating CASC15-S increased cellular growth and migratory capacity. Gene expression analysis revealed downregulation of neuroblastoma-specific markers in cells with attenuated CASC15-S, with concomitant increases in cell adhesion and extracellular matrix transcripts. Altogether, our results point to CASC15-S as a mediator of neural growth and differentiation, which impacts neuroblastoma initiation and progression.

Figure 1

Fine mapping of 6p22 identifies CASC15-S as a candidate cis-acting neuroblastoma susceptibility gene. (a) Regional association plot of single nucleotide polymorphisms (SNPs) generated with LocusZoom software using genome wide imputation data from 2,817 neuroblastoma cases and 7,473 controls. The pair-wise linkage disequilibrium (r²) for each SNP is denoted by color, and the log-transformed p-values for each SNP are shown on the y-axis. Thirty-two SNPs with highly significant p-values (< 1×10⁻¹⁰) ranging from 4.67×10⁻¹⁰ to 4.81×10⁻¹⁷ were found to cluster as a narrow peak within a genomic region containing the lncRNAs CASC14 and CASC15. (The exons and transcribed regions of genes are shown as solid vertical lines.) (b) The haplotype structure of this region in Northern European (CEU) population demonstrates a signal that overlaps with these SNPs, refining and initial 94.2kb LD block (boundaries denoted by black vertical dashed lines) down to 34.9kb, demonstrated by the dashed red vertical line and rightmost black vertical dashed line. (SNP annotations: color = representative of LD r² value (purple = reference SNP); predicted coding or 3’UTR region; = nonsynonymous; * = tfbccons, conserved motif at transcription factor binding site, = mcs44placental, highly conserved region in placental mammals).

Figure 2

CASC15-S is the predominant lncRNA isoform expressed in neuroblastoma. (a) Graphical representation of the lncRNA transcripts observed to originate from the 6p22.3 locus via RNA sequencing. This locus includes two predominant transcripts on the positive strand: a 1.9kb CASC15 transcript (current RefSeq for this lncRNA) and a novel shorter (1.2kb) transcript, CASC15-S that shares the last 3 exons with the long isoform of CASC15. These transcripts were validated using 5’ and 3’ RACE. Epigenetic marks support active transcription (RNA PolII occupancy) and an enhancer like function (H3K27Ac, H3K4Me3) of this region, including a proximal VISTA enhancer element (hs1335). RNA sequencing, RNA PolII, DNAse, and P300 ChIP-Seq data were obtained from SK-N-SH neuroblastoma cells (via ENCODE), whereas enhancer track ChIP-Seq data was taken from MGG8 human glioblastoma stem cells . (b)Expression levels of CASC15, CASC15-S, CASC14 and hs1335 were normalized and quantified across a panel of 16 normal primary tissues (Human Body Map 2.0 project), indicating predominant expression of the CASC15-S isoform in brain. (c) RNA sequencing performed on 108 primary neuroblastoma tumors, analyzed for unique isoform expression, exhibited a predominance of the short CASC15 isoform. This short isoform was expressed at 20-fold and 40-fold higher levels than CASC14 and or full-length CASC15, respectively. (d) RNA fluorescent in-situ hybridization (RNA-FISH) was conducted in NGP neuroblastoma cells for several transcripts known to exist at this locus. To ensure probe specificity, odd and even pools of fluorescently labeled oligonucleotide probes were used to tile CASC15-S and VISTA hs1335. The yellow fluorescence observed in the “merge” panel was obtained from overlap of the hs1335 and CASC15-S fluorescent signals and indicate colocalization of these two transcripts. Consistent with the majority of described lncRNAs, these transcripts appear to be predominately nuclear as evidenced in the “merge + DAPI” panel. (40× magnification)

Figure 3

Identification of rs9295534 as a functional polymorphism. (a) Filtration strategy employed to refine list of imputed SPSs at the 6p22.3 locus for functional variants. We identified SNPs with robust p-values (< 1×10⁻¹⁰) that fell within regions of regulator chromatin (DNase hypersensitivity) and putative enhancer activity (H3K27 acetylation) in SK-N-SH neuroblastoma cells. This resulted in four highly significant polymorphisms: rs1543310, rs6905441, rs9295534 and rs9368402. Further refinement based on evolutionary conservation identified rs9295534 as the only SNP to fit all criteria. (A full table of SNP attributes is given in Supplementary Table 1) (b) Evidence from glioblastoma cells revealing that rs9295534 (light blue vertical line) maps to the most proximal enhancer site to CASC15-S as evidenced by DNaseI sensitivity (DS), H3K4me1 and H3K27ac marks. (c) Further characterization of rs9295534 was indicated due to a robust p-value (p=3.51×10⁻¹²), as well as the inclusion of this SNP within a region of predicted enhancer activity (H3K27Ac peaks) in several fetal tissues, including fetal adrenal gland. (d) Functional demonstration of rs9295534 was accomplished by insertion of 1500bp risk (A_A) or non-risk (T_T) fragments (cloned from Lan5 and Chp134 neuroblastoma cell lines, respectively) into a luciferase reporter vector (pGL4.23) upstream of a minimal promoter. Luciferase activity was normalized to a contransfected Renilla luciferase vector (pGL4.75). Expression of the risk fragment resulted in significantly attenuated transcriptional activity than was observed with the non-risk fragment following quantification of luciferase activity in HEK-293 cells (p < 0.0001).

Figure 4

Low CASC15-S expression correlates with poor clinical prognosis. (a) Microarray expression data from clinically annotated primary neuroblastoma tumors (n=250) obtained at diagnosis reveals that high-risk (stage 4) neuroblastomas (n=220) demonstrate significantly lower expression of CASC15-S than low-risk (stage 1) tumors (n=30). (b) Kaplan-Meier analysis demonstrates significantly poorer overall survival for children with tumors expressing low levels of CASC15-S (n=163 for group “low”, n=87 for group “high”, adj. p = 3.2×10⁻⁰⁶). (c) Relevant metrics for selection of high-risk patient tumors used for differential gene expression analyses between patients with high (n=74) and low (n=146) CASC15-S levels. CASC15-S was expression was increased 1.9-fold in the “High” group, and these patients exhibited a significant increase in overall survival. (d) The most highly significantly regulated pathway using GSEA was identified to be a set of genes downregulated in poor outcome neuroblastoma (Asgharzadeh neuroblastoma poor survival down). Patients with high CASC15-S expression demonstrated enrichment of these genes, suggesting that CASC15-S acts in a protective manner. (*** p < 0.0001).

Figure 5

CASC15-S depletion induces a more aggressive phenotype in neuroblastoma cells. (a) CASC15-S expression was investigated by qRT-PCR in a panel of neuroblastoma cell lines (n=21) and normalized relative to the geometric mean of GUSB, HPRT and TBP housekeeping genes. CASC15-S demonstrated a wide range of expression across neuroblastoma cell lines. (b) SK-N-BE2 neuroblastoma cells transiently transfected with siRNA targeting an exon common to both CASC15 and CASC15-S isoforms (exon 12 or exon 4, respectively), or (c) specifically targeting only the unique exon of CASC15-S (exon 1), show a significant increase in proliferative rate. (d) Stable depletion of CASC15-S in SK-N-BE2 cells was achieved with lentiviral transduction of shRNA, and recapitulated the increased growth observed with transient knockdown. (e) Forced ectopic expression of CASC15-S cDNA was able to rescue the growth characteristics of SK-N-BE2 shCASC15-S cells, reverting their growth pattern to that of wild-type cells (f) Morphological observation of SK-N-BE2 cells stably depleted of CASC15-S showed that cells were substantially larger than control cells (scale bar = 100μm). (g) Cell area was measured in biological triplicate (n=10 for each replicate) and quantified in ImageJ, where the area of shCASC15 cells was found to be 3.1-fold increased over controls (*** p < 0.001). siNTC = non-targeting control (negative control), siPLK1 = polo-like kinase 1 (positive control)

Figure 6

CASC15-S regulates a subset of genes involved in neural differentiation and neuroblastoma tumorigenesis. (a) SK-N-BE2 cells constitutively depleted of CASC15-S demonstrated an increased migratory capacity in wound healing assays (t=24h). Linear regression comparison of wound closure at regular intervals demonstrates a clear enhancement of migration in silenced SK-N-BE2 (b) or SK-N-SH (c) cells (EV = Empty Vector). (d) Gene ontology (GO) analysis of SK-N-SH cells following CASC15-S depletion via siRNA at 48 hours. The top-level biological processes are shown by percentage of enrichment signal, with both locomotion and cellular adhesion gene sets exhibiting over representation. (e) A similar analysis was carried out for SK-N-BE2 cells stably expressing an shRNA construct targeting CASC15-S. The top enrichment observed in these cells, “cellular process” was primarily the result of the cell differentiation gene subset. (f) Most significantly altered pathways in SK-N-SH and SK-N-BE2 neuroblastoma cells following depletion of CASC15-S and subjected to Ingenuity pathway analysis. Pathways shown were the top gene signatures to arise from differential analysis, and indicate activation of cellular programs of proliferation, migration and metastasis (shown in red), as well as downregulation of several pathways known to modulate neural-specific development (shown in green).