Functional MYCN signature predicts outcome of neuroblastoma irrespective of MYCN amplification

Abstract

Neuroblastoma is a pediatric tumor of the sympathetic nervous system. MYCN (V-myc myelocytomatosis viral-related oncogene, neuroblastoma derived [avian]) is amplified in 20% of neuroblastomas, and these tumors carry a poor prognosis. However, tumors without MYCN amplification also may have a poor outcome. Here, we identified downstream targets of MYCN by shRNA-mediated silencing MYCN in neuroblastoma cells. From these targets, 157 genes showed an expression profile correlating with MYCN mRNA levels in NB88, a series of 88 neuroblastoma tumors, and therefore represent in vivo relevant MYCN pathway genes. This 157-gene signature identified very poor prognosis tumors in NB88 and independent neuroblastoma cohorts and was more powerful than MYCN amplification or MYCN expression alone. Remarkably, this signature also identified poor outcome of a group of tumors without MYCN amplification. Most of these tumors have low MYCN mRNA levels but high nuclear MYCN protein levels, suggesting stabilization of MYCN at the protein level. One tumor has an MYC amplification and high MYC expression. Chip-on-chip analyses showed that most genes in this signature are directly regulated by MYCN. MYCN induces genes functioning in cell cycle and DNA repair while repressing neuronal differentiation genes. The functional MYCN-157 signature recognizes classical neuroblastoma with MYCN amplification, as well as a newly identified group marked by MYCN protein stabilization.

Fig. 1.

Gene regulation by MYCN. (A) Overview of R2 bioinformatics tools used to identify MYCN-regulated genes relevant to neuroblastoma. In the Time Series module, genes are selected for regulation by MYCN. Correlation with MYCN in neuroblastoma tumors (NB88) reduces the group to 157 regulated genes relevant to tumors. These genes are analyzed individually or as a group (gene category: MYCN-157) with the different tools indicated on the right. R2 programs are indicated with boxes, and tool names are in italics. (B–E) PRMT1 is regulated by MYCN. (B) IMR32 was transduced with shMYCN (blue lines) or control virus (gray line). The expression graph shows the PRMT1 mRNA expression of the Affymetrix profiling. (C) ChIP-on-chip analysis in IMR32 with anti-MYCN or control antibody. Signal: raw data of binding ratios (²logfold) in red bars. Score: the significant binding region (²logfold) (FDR: < 0.05). The three reference PRMT1 transcripts (exon dark, intron light green) are aligned to their chromosomal position, and MYCN consensus sites are indicated below (CACGTG in red and the alternative site in green; Dataset S1). (D) YY plot showing the correlation of MYCN (red) and PRMT1 (blue) in neuroblastoma tumor series NB88. Below the graph tracks for survival, MYCN amplification and International Neuroblastoma Staging System stages are shown. (E) Overall survival for PRMT1 expression in the NB88 set.

Fig. 2.

The expression of MYCN-157 signature predicts clinical outcome of neuroblastoma. Overall survival analysis was performed in tumor series NB88 (A and B) and NB251 (C and D). The gene expression of MYCN-157 in neuroblastoma tumors was clustered with K-means, resulting in two groups: MYCN-157-POS and MYCN-157-NEG (see Fig. S3 for NB88). Overall survival is shown for the clustering according to MYCN-157 with (A and C) and without (B and D) MYCN-amplified (MNA) tumors.

Fig. 3.

MYC amplification and high MYCN protein expression in neuroblastoma tumors clustering together with MYCN-amplified tumors. (A) MYCN and MYC mRNA expression detected by Affymetrix profiling in MYCN-157-NEG and MYCN-157-POS tumors. The MYCN-157-POS group was divided by absence or presence of MYCN amplification (MNA). (B) Southern blot of tumor (T) and normal (N) DNA of patient itcc0184 showing MYC amplification. The control probe is L.2.3 (29). (C) MYCN protein expression analysis on tissue array compared with MYCN mRNA expression. Tumors were scored for expression of nuclear MYCN according to the following categories of expression: none (0), weak (1), moderate (2), or strong (3). The y axis shows the expression of MYCN mRNA. MYCN-amplified tumors are indicated in red and nonamplified tumors in blue.

Fig. 4.

MYCN binds preferentially to up-regulated genes. (A) ChIP-on-chip analysis of MYCN and control in IMR32. The percentage of significant (FDR < 0.05) bound promoters at the TSS (TSS-1) and ± 2,000 bp (TSS-2000) is shown. (B) The percentage of genes with a CACGTG ± 100 bp (TSS-100) or ± 2,000 bp (TSS-2000) is shown. The analysis was performed for MYCN-157 (regulated: yes), the up-and down-regulated subsets, and all other genes (regulated: no). *Significant association (P < 0.01) between bound/unbound genes or presence/absence of CACGTG and regulation in the MYCN-157 sets (two-sided Fisher’s exact test).

Fig. 5.

DNA repair and neuronal differentiation genes are highly expressed in neuroblastoma tumors. MegaSampler analysis in normal and tumor tissues. The box plots show the expression of DNA repair genes (A) and neuronal differentiation genes (B) in neuroblastoma and normal tissue. Normal tissues [green (30)] are divided in the 10 groups indicated below. Four independent neuroblastoma series [red: NB30 (31), NB51, NB64 (32), and NB88] are named after the sample number in the group.