Impact of Interleukin-6 –174 G>C Gene Promoter Polymorphism on Neuroblastoma

<div><p>Background</p><p>Common variants in DNA may predispose to onset and progression of neuroblastoma (NB). The genotype GG of single nucleotide polymorphism (SNP) rs1800795 (−174 G>C) in interleukin (IL)-6 promoter has been associated with lower survival of high-risk NB.</p><p>Result</p><p>To evaluate the impact of <i>IL-6</i> SNP rs1800795 on disease risk and phenotype, we analyzed 326 Italian NB patients and 511 controls. Moreover, we performed <i>in silico</i> and quantitative Real Time (qRT)-PCR analyses to evaluate the influence of the SNP on gene expression in 198 lymphoblastoid cell lines (LCLs) and in 31 NB tumors, respectively. Kaplan-Meier analysis was used to verify the association between <i>IL-6</i> gene expression and patient survival. We found that <i>IL-6</i> SNP is not involved in susceptibility to NB development. However, our results show that a low frequency of genotype CC is significantly associated with a low overall survival, advanced stage, and high-risk phenotype. The <i>in silico</i> (<i>p</i> = 2.61×10⁻⁵) and qRT-PCR (<i>p</i> = 0.03) analyses showed similar trend indicating that the CC genotype is correlated with increased level of <i>IL-6</i> expression. In report gene assay, we showed that the −174 C variant had a significantly increased transcriptional activity compared with G allele (<i>p</i> = 0.0006). Moreover, Kaplan-Meier analysis demonstrated that high levels of <i>IL-6</i> are associated with poor outcome in children with NB in two independent gene expression array datasets.</p><p>Conclusions</p><p>The biological effect of SNP <i>IL-6</i>–174 G>C in relation to promotion of cancer progression is consistent with the observed decreased survival time. The present study suggests that SNP <i>IL-6</i>–174 G>C may be a useful marker for NB prognosis.</p></div

Case-Control study of <i>IL-6</i>–176 (G>C) SNP.

a<p>P-value and OR obtained by Armitage's trend test.</p

Kaplan-Meier curves for OS rates.

<p>OS rates were compared between CC and any G (GC or GG) for the SNP <i>IL-6</i>–174 in (A) all patients, (B) in not <i>MYCN</i> amplified patients, (C) in high-risk patients and (D) not <i>MYCN</i> amplified high-risk patients.</p

Characteristics of NB patients stratified per <i>IL-6</i> -176 (G>C) SNP genotype.

<p>N.A.  =  not available.</p>a<p>p-values and ORs from comparison of allelic frequencies.</p>b<p>p-values and ORs from comparison of genotype frequencies (GG/GC vs CC).</p>c<p>p-values and ORs from comparison of stage 1+2 patients vs stage 3+4 patients.</p

Test for independent statistical significance of −174 <i>IL-6</i> SNP after adjustment for NB risk factors.

<p>HR, hazard ratio; CI, confidence interval; C-index, concordance index.</p>a<p>C-index calculated including in the model only the NB risk factor (Age or MYCN or INSS stage 4).</p>b<p>C-index calculated including in the model the NB risk factor and <i>IL-6</i> SNP.</p

SNP genotype correlation to <i>IL-6</i> gene expression and NB outcome.

<p><i>In silico</i> and qRT-PCR analysis of <i>IL-6</i> mRNA expression in (A) 198 LCLs and (B) 31 NB tumors, respectively, stratified according to the SNP <i>IL-6</i>–174. (C) Luciferase report gene assay carried out in HEK293 cells. Data shown in percentage are the mean ± SD from three independent transfection experiments, each done in triplicate and compared with promoter less control. Kaplan-Meier analysis is shown, with individuals grouped by median of expression of <i>IL-6</i> for OS and Progression Free Survival (PFS) rates in (D and E) 88 NB patients and (F) only PFS for 102 INSS stage 4 patients with <i>MYCN</i> not amplified. OS data of Seeger dataset were not available.</p

The four-gene signature predict neuroblastoma outcome.

<p>Kaplan–Meier and log-rank analysis for progression-free (PFS) and overall (OS) survival in discovery set 1 (A), set 3 (B), and set 4 (C) based on the four-gene signature score. (D, E) Comparison of signature performance between all 25 and the 4 selected genes. Results from discovery and validation study strategy. (D) Gene predictors made in discovery set 4 and predictions made in discovery set 1. (E) Gene predictors made in validation set 4 and predictions made in discovery set 5. X axis indicates clinical factors: death event (DE), relapse event (RE), and INSS stage; Y axis indicates Area Under Curve (AUC).</p

MDM2 inhibition stabilizes p53 and induces apoptosis and p53 signaling pathways in neuroblastoma cells.

<p>(A) Consistent with our previously data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079843#pone.0079843-Barbieri1" target="_blank">[11]</a>, Western blotting showed an increase in p53 levels within 4 hours exposure to Nutlin-3a in p202, p218, and IMR32 cells, confirming that the p53 signaling pathway is active in our system. (B) Apoptosis induction was tested in five established neuroblastoma lines (JF, IMR32), early passage neuroblastoma tumor cultures (p202, p218, H), and four non-neuroblastoma human solid tumors including colorectal (HCT116), breast (MCF7) and osteosarcoma (SJSA-1). SJSA-1 line differs by the level of MDM2 expression, being amplified 25 fold. A p53 mutant neuroblastoma line (SJ3-12), which lacks part of the DNA binding domain, was used as control. Proliferating cells were treated with Nutlin-3a for 24 hours and TdT-positive fraction was measured by flow cytometry. (C) Pathways enriched upon Nutlin-3a treatment: p53 signaling (GSEA ID: M6370), DNA damage (GSEA ID: M8378), and chemotherapy response (bleo_human_lymph) genes. Vertical columns separate Nutlin-3a versus Nutlin-3b at 3, 8, and 16 hours, horizontal rows indicate genes.</p

Study design.

<p>Freshly isolated GD2 positive cells were obtained by positive immunomagnetic bead manipulation of BM aspirates, as described in M&M section. Primary NB tumors were stored in the Tissue Repository at the Gaslini Institute.</p

The four-gene signature is regulated by p53 activity.

<p>Activation of p53 represses the four-gene signature. (A, B) Quantitative PCR demonstrates marked decrease of mRNA levels of the four genes after Nutlin-3a treatment (10 uM for 8 hours) in multiple p53 wild-type neuroblastoma lines (shown here LAN5 and IMR32). However, this effect is completely abrogated after p53 silencing or when the effect of Nutlin-3a is tested in a p53 mutant neuroblastoma cell line (LAN1) (C). Each error bar represents two biological replicates.</p