Regulation of MYCN expression in human neuroblastoma cells

Background Amplification of the MYCN gene in neuroblastoma (NB) is associated with a poor prognosis. However, MYCN-amplification does not automatically result in higher expression of MYCN in children with NB. We hypothesized that the discrepancy between MYCN gene expression and prognosis in these children might be explained by the expression of either MYCN-opposite strand (MYCNOS) or the shortened MYCN-isoform (ΔMYCN) that was recently identified in fetal tissues. Both MYCNOS and ΔMYCN are potential inhibitors of MYCN either at the mRNA or at the protein level. Methods Expression of MYCN, MYCNOS and ΔMYCN was measured in human NB tissues of different stages. Transcript levels were quantified using a real-time reverse transcriptase polymerase chain reaction assay (QPCR). In addition, relative expression of these three transcripts was compared to the number of MYCN copies, which was determined by genomic real-time PCR (gQPCR). Results Both ΔMYCN and MYCNOS are expressed in all NBs examined. In NBs with MYCN-amplification, these transcripts are significantly higher expressed. The ratio of MYCN:ΔMYCN expression was identical in all tested NBs. This indicates that ΔMYCN and MYCN are co-regulated, which suggests that ΔMYCN is not a regulator of MYCN in NB. However, the ratio of MYCNOS:MYCN expression is directly correlated with NB disease stage (p = 0.007). In the more advanced NB stages and NBs with MYCN-amplification, relatively more MYCNOS is present as compared to MYCN. Expression of the antisense gene MYCNOS might be relevant to the progression of NB, potentially by directly inhibiting MYCN transcription by transcriptional interference at the DNA level. Conclusion The MYCNOS:MYCN-ratio in NBs is significantly correlated with both MYCN-amplification and NB-stage. Our data indicate that in NB, MYCN expression levels might be influenced by MYCNOS but not by ΔMYCN.


Background
Neuroblastoma cells (NBs) that carry an amplified MYCN gene are extremely malignant. However, MYCN-amplification does not automatically result in higher expression of MYCN in children with NB [1][2][3]. Thus, it has been suggested that the aggressive phenotype of MYCN amplified NBs may be explained by higher expression levels of other genes co-amplified with MYCN, since the amplified unit of DNA can be up to 1 Mb. To date, three genes have been identified that are frequently co-amplified with MYCN in NBs: DDX1 in 50% of the cases, NAG in 20% of the tumours, and MYCNOS in all cases [4,5]. All three genes demonstrate increased transcript expression when coamplified in NB cell lines, indicating that they may contribute to tumour phenotype. However, survival analyses in a large study using 75 MYCN-amplified tumours indicate that neither amplified DDX1 nor NAG have an additional adverse effect on the prognosis of the patients [6].
Natural antisense transcripts are abundant in eukaryotic genomes [7][8][9]. In human, more than 1600 natural antisense transcript are predicted to be present [10]. They can influence gene expression on the DNA level by transcriptional interference, on the transcript level by RNA interference and RNA editing, or direct splicing by RNA masking [11,12]. MYCNOS is the antisense transcript of MYCN [13] and shows overlap with the first exon of MYCN. This antisense transcript could be involved in modulating the expression of MYCN by any of the mechanisms mentioned above. Antisense transcripts are considered to be relevant to the development and progression of tumours [14][15][16], but until now, only antisense HIF-1α RNA has been shown to be a marker for prognosis in human breast cancer [17].
Recently, we reported a fetal MYCN splice variant (ΔMYCN) lacking exon 2 [18]. The ΔMYCN transcript is expressed in several fetal tissues and contains the acidic region, nuclear localization signal, the basic helix-loophelix and leucine-zipper domains but lacks the transactivation domain. It has been suggested that the ΔMYCN protein may serve as an obligate dimerization partner for MYCN to convey transcriptional activation or repression.
In this report we analysed whether expression of ΔMYCN and MYCNOS influence MYCN expression levels in NBs of different disease stages.

Tumour material
Sixteen fresh-frozen NBs were obtained from at the Department of Pathology at the Radboud University Nijmegen Medical Centre. All NBs were derived from pediatric patients (0 to 6-years-old) diagnosed at the Department of Pediatric Hemato-Oncology. Sections of the frozen samples were stained with hematoxylin-eosin and reviewed by the pathologist to verify tumour histology and to evaluate the percentage of tumour cells. Samples were only considered for study if the contents of tumour cells was ≥ 75%. Six out of 16 NBs had MYCNamplification as shown by southern blot and/or fluorescent in situ hybridization (FISH). All samples were anonymized prior to this study, and the research program was approved by the local ethics committee (Commissie Mensgebonden Onderzoek Regio Arnhem-Nijmegen).

DNA isolation, RNA isolation and cDNA synthesis
Tumour samples were aliquoted in two parts to isolate both DNA and RNA. Total DNA was isolated with the QIAamp isolation-kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. All samples were RNAse treated. Total RNA was isolated with TriZol reagent (Invitrogen, Carlsbad, CA, USA) and treated with Deoxyribonuclease I (Dnase I; Invitrogen). DNase-treated RNA was reverse-transcribed using oligo(dT) primers with the SuperScript First-Strand Synthesis System (Invitrogen).

PCR
MYCN and splice variants were amplified from cDNA by using the GC-RICH PCR System (Roche Applied Science, Almere, The Netherlands). Primers were developed by the primer3 program (http://frodo.wi.mit.edu/cgi-bin/ primer3/primer3_www.cgi [19]). Primer sequences are shown in table 1 and the position of the primers in MYCN are depicted in figure 1. PCR products were sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing V2.0 Ready Reaction Kit and analysed with the ABI PRISM 3730 DNA analyser (Applied Biosystems, Foster City, CA, USA).

QPCR and gQPCR
QPCR and gQPCR was performed by SYBR Green-based quantification (Bio-Rad, Veenendaal, The Netherlands). PCRs were performed on an iCycler (MyiQ single color Real-Time detection System, Bio-Rad, Veenendaal, The Netherlands). Sequences of the primers used to quantify cDNA transcript levels and genomic DNA are shown in table 1 and the position of the primers is depicted in figure 1. PCR products were between 80-and 100-bp. Validation of the primer pairs and (g)QPCR experiments were performed as described previously [20,21]. Differences in expression of a gene of interest or in genomic DNA copy number between two samples were calculated by the comparative Ct or 2 ΔΔCt method [22,23]. Hoebeeck et al. described and validated a similar assay for the determination of MYCN copy numbers in tumor samples [24].
For immunoprecipitation, 5 μg C-19 mAb (Santa Cruz Biotechnology, Heidelberg, Germany) was coupled to Prot A sepharose CL-4B beads (Pharmacia Biotechnologies, Uppsala, Sweden) for 1 hour at 4°C to). NB-lysates were precleared O/N with 50 μl packed Prot A sepharose CL-4B beads. To the precleared lysates, 20 μl C-19-coupled beads was added and incubated for 24 h at 4°C. Subsequently, the beads were washed with PBS and resuspended in SDS sample buffer and stored at -80°C until SDS-polyacrylamide gel electrophoresis (PAGE).

SDS-PAGE and Western blotting
Samples (30 μg homogenized NB-sample or 20 μl precipitation beads) were separated a 10% polyacrylamide gels and transferred to nitrocellulose (Hi-bond, Amersham Biosciences, Little Chalfont, UK). Ponceau S staining was used to confirm that equal amounts of protein were loaded in each lane (additional file 1). The nitrocellulose blot was blocked with 1% BSA in 20 mM Tris-HCl pH 7.4 and 0.1% Tween (Tris-buffered saline/Tween 20; TBS-T) for 1 h. The blot was washed for 5 min with TBS-T followed by incubation with the C-19 anti-MYCN antibody 1:200 diluted in TBS-T for 1 h at RT. After washing 3× with TBS-T, the blot was incubated with HRP-conjugated swine-anti-rabbit antibody (1:5000 diluted in TBS-T) for 1 h at RT. Subsequently, the blot was washed and incubated for 1 min with ECL substrate (Amersham Biosciences, Little Chalfont, UK) and exposed to film (Kodak, Rochester, NY, USA).

Overexpression of MYCNOS in the NB cell line IMR-32
Primers for the amplification of MYCNOS were developed by using the primer3 program (Table 1). MYCNOS was amplified from DNA isolated from a healthy control using the GC RICH PCR System (Roche, Woerden, The Netherlands). Subsequently, MYCNOS was cloned into the Gateway donor vector pDONR-201 (Invitrogen). Using the Gateway cloning system, MYCNOS was subsequently subcloned into the pcDNA3 expression vector and integrity of the construct was validated by sequence analysis. IMR-32 NB cells at 50% confluence in a 25 cm 2 flask were cotransfected with 8 μg pcDNA3-MYCNOS and C1-GFP using 120 μl lipofectamine (Invitrogen) in 3 ml Opti-MEM-I for 20 min at RT.

Patient characteristics
We analysed fresh-frozen NBs from 16 pediatric patients (age range: 0-6 years old). The NBs are classified according to the Children's Cancer Group Neuroblastoma Staging System [25] and treated according to Pediatric Oncology Group-protocols (

ΔMYCN expression in the neuroblastoma cell line IMR-32
Two splice variants have been described for the protooncogene MYCN, the classical transcript that consists of three exons and a shortened ΔMYCN transcript that lacks exon 2. ΔMYCN is expressed in several fetal tissues, but its expression has not been reported in NBs. We used primers spanning exon 2 ( Figure 1A and and MYCNOS are also significantly increased in NBs with MYCN-amplification (p < 0.01; Figure 3A).

Correlation of NB stage with MYCN:ΔMYCN-ratio showed that the MYCN:ΔMYCN-ratio remains constant and does not change with either MYCN-amplification
Detection of MYCN and ΔMYCN in IMR-32 cells      Figure 3C; two-tailed p-value = 0.09, calculated with the Students' T-test) or NB stage ( Figure 3F; two-tailed p-value = 0.12, calculated with the Spearman rank correlation). However, there is a significant correlation between the MYCN:MYCNOS-ratio and both MYCN-amplification ( Figure 3C; two-tailed p-value = 0.025) and NB-stage (Figure 3E; two-tailed p-value = 0.007). The MYC-NOS:ΔMYCN-ratio did not significantly change with either MYCN-amplification or NB-stage (two-tailed p-values = 0.58 and 0.24, respectively; data not shown). These data show that in more advanced NB tumours, mRNA expression of MYCNOS increases relative to MYCN.

MYCN-amplification
To more exactly determine MYCN copy number in the tumours that were studied, we performed a genomic quantitative PCR (gQPCR) using genomic primers recognizing five different locations within the MYCN-gene (Figure 1A; Table 1). Amplification of these DNA fragments was calculated relative to three reference genes elsewhere on the genome, CFTR, TBX22, and SLC16A2. Among these three reference genes, there were no copy number differences noted in any of the NB samples. All 10 samples with a normal MYCN-copy number based on Southern blotting and/or FISH, carried two to four MYCN copies as determined by gQPCR. The presence of a MYCN duplication in NB cells that lack an overt amplification of MYCN is more often found, although the implications for the progression of the NB are still unclear [5,27]. All 6 samples with multiple copies of MYCN, as determined by Southern blotting and/or FISH, had in between 20 and 139 MYCN gene amplifications (Table 2), which is within the normal range of gene copy numbers observed in NBs with MYCN-amplification [28].
We observed that MYCN mRNA-expression does not linearly correlate with MYCN-amplification, consistent with earlier reports [26]. In addition, also ΔMYCN and MYC-NOS do not correlate linearly with the number of MYCN gene copies ( Figure 3B). As shown in figure 3D, the relative mRNA expression ratio of MYCN:MYCNOS decreases with an increasing number of MYCN gene copies although this is not significant (non-interrupted line, slope = -0.4; two-tailed p-value = 0.18, calculated with the Pearson correlation test). The MYCN:ΔMYCN ratio does not change with higher MYCN copy numbers ( Figure 3D; dotted line, slope = -0.1; two-tailed p-value = 0.88).

Overexpression of MYCNOS in the NB cell line IMR-32
The pre-mRNA of MYCNOS, which represents the MYCN antisense transcript, shows overlap with the first exon of MYCN. Therefore MYCNOS may potentially modulate MYCN mRNA expression levels at the mRNA level via RNA-interference or RNA-editing, or direct MYCN splic-ing by RNA masking [11]. To test this premise, we transfected IMR-32 NB cells with C1-GFP and either the pcDNA3-vector containing MYCNOS or an empty vector.

Discussion
In this report, we have analysed the expression levels of Besides MYCN and MYCNOS, ΔMYCN, which was previously identified as a fetal transcript [18], is also expressed in NBs. No other MYCN isoforms were detected. This suggests that the alternative splice variant that previously has been described by Stanton et al. [30] and consists of an alternatively spliced exon 1, has little relevance in the progression of NBs. In one tumour, the ΔMYCN transcript could not be identified, but this is probably because ΔMYCN is low expressed in general and in this tumour leucine-zipper domain, which may serve to dimerize with MYCN or bind to its DNA binding site. ΔMYCN lacks the transactivation domain including the highly conserved Myc 1 and 2 boxes, from which it was speculated that it competes with MYCN and therefore inhibits the active MYCN protein [18]. However, in all neuroblastoma samples analyzed, the ratio between MYCN and ΔMYCN expression remains constant and does not correlate with MYCN-amplification or disease stage, indicating that ΔMYCN induced inhibition of MYCN at the protein level is not of relevance in NB.

Conclusion
In conclusion, our results suggest that the expression of the antisense gene MYCNOS might be relevant to the progression of NB, potentially by directly inhibiting MYCN transcription by transcriptional interference at the DNA level. Analysis of MYCN:MYCNOS expression ratios in patients with NB without MYCN-amplification and clinical follow-up are necessary to establish the relevance of MYCNOS expression to the prognosis of these patients.