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      Conditional expression of retrovirally delivered anti-MYCN shRNA as an in vitro model system to study neuronal differentiation in MYCN-amplified neuroblastoma

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          Abstract

          Background

          Neuroblastoma is a childhood cancer derived from immature cells of the sympathetic nervous system. The disease is clinically heterogeneous, ranging from neuronal differentiated benign ganglioneuromas to aggressive metastatic tumours with poor prognosis. Amplification of the MYCN oncogene is a well established poor prognostic factor found in up to 40% of high risk neuroblastomas.

          Using neuroblastoma cell lines to study neuronal differentiation in vitro is now well established. Several protocols, including exposure to various agents and growth factors, will differentiate neuroblastoma cell lines into neuron-like cells. These cells are characterized by a neuronal morphology with long extensively branched neurites and expression of several neurospecific markers.

          Results

          In this study we use retrovirally delivered inducible short-hairpin RNA (shRNA) modules to knock down MYCN expression in MYCN-amplified (MNA) neuroblastoma cell lines. By addition of the inducer doxycycline, we show that the Kelly and SK-N-BE(2) neuroblastoma cell lines efficiently differentiate into neuron-like cells with an extensive network of neurites. These cells are further characterized by increased expression of the neuronal differentiation markers NFL and GAP43. In addition, we show that induced expression of retrovirally delivered anti- MYCN shRNA inhibits cell proliferation by increasing the fraction of MNA neuroblastoma cells in the G1 phase of the cell cycle and that the clonogenic growth potential of these cells was also dramatically reduced.

          Conclusion

          We have developed an efficient MYCN-knockdown in vitro model system to study neuronal differentiation in MNA neuroblastomas.

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          Most cited references30

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          Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells.

          RNA interference (RNAi) was first recognized in Caenorhabditis elegans as a biological response to exogenous double-stranded RNA (dsRNA), which induces sequence-specific gene silencing. RNAi represents a conserved regulatory motif, which is present in a wide range of eukaryotic organisms. Recently, we and others have shown that endogenously encoded triggers of gene silencing act through elements of the RNAi machinery to regulate the expression of protein-coding genes. These small temporal RNAs (stRNAs) are transcribed as short hairpin precursors (approximately 70 nt), processed into active, 21-nt RNAs by Dicer, and recognize target mRNAs via base-pairing interactions. Here, we show that short hairpin RNAs (shRNAs) can be engineered to suppress the expression of desired genes in cultured Drosophila and mammalian cells. shRNAs can be synthesized exogenously or can be transcribed from RNA polymerase III promoters in vivo, thus permitting the construction of continuous cell lines or transgenic animals in which RNAi enforces stable and heritable gene silencing.
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            RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells.

            Duplexes of 21-nt RNAs, known as short-interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference (RNAi) when introduced into mammalian cells. We show that siRNAs can be synthesized by in vitro transcription with T7 RNA polymerase, providing an economical alternative to chemical synthesis of siRNAs. By using this method, we show that short hairpin siRNAs can function like siRNA duplexes to inhibit gene expression in a sequence-specific manner. Further, we find that hairpin siRNAs or siRNAs expressed from an RNA polymerase III vector based on the mouse U6 RNA promoter can effectively inhibit gene expression in mammalian cells. U6-driven hairpin siRNAs dramatically reduced the expression of a neuron-specific beta-tubulin protein during the neuronal differentiation of mouse P19 cells, demonstrating that this approach should be useful for studies of differentiation and neurogenesis. We also observe that mismatches within hairpin siRNAs can increase the strand selectivity of a hairpin siRNA, which may reduce self-targeting of vectors expressing siRNAs. Use of hairpin siRNA expression vectors for RNAi should provide a rapid and versatile method for assessing gene function in mammalian cells, and may have applications in gene therapy.
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              Distinct transcriptional MYCN/c-MYC activities are associated with spontaneous regression or malignant progression in neuroblastomas

              Background Neuroblastoma is the most common extracranial malignant solid tumor in early childhood. Clinical courses are highly variable, ranging from spontaneous regression to therapy-resistant progression. Clinical and biological features, such as age at diagnosis, disease stage, numerical (ploidy) and structural chromosomal alterations (MYCN gene amplification; 1p, 3p, 11q deletions; 17q gain), are associated with patient outcome [1,2]. Amplified MYCN oncogene identifies a subtype with poor prognosis [3] and is consistently associated with high MYCN mRNA and protein levels. There is strong experimental evidence (ectopic MYCN expression in cell lines, N-myc transgenic neuroblastoma mouse model) that increased MYCN activity is involved in tumor initiation and progression of at least a subset of neuroblastomas [4,5]. The MYC gene family members, c-MYC, MYCN and MYCL, are involved in the biology of many cancer types. They encode basic helix-loop-helix leucine zipper proteins that are found as heterodimers with their obligate partner protein, MAX [6]. The MYC-MAX heterodimer binds to DNA consensus core binding sites, 5'-CACGTG-3' or variants thereof (E-boxes), which preferentially leads to transcriptional activation of target genes. Repression of target genes by MYC proteins has also been described [7]. This seems to be independent of the binding of MYC proteins to E-boxes, but involves a cofactor, Miz-1, that tethers MYC-MAX to gene promoters, such as p15 and p21. Enhanced activity of MYC transcription factors contributes to almost every aspect of tumor formation: unrestricted cell proliferation, inhibition of differentiation, cell growth, angiogenesis, reduced cell adhesion, metastasis, and genomic instability [6,8]. In contrast, MYC transcription factors, including MYCN, also sensitize cells for apoptosis, a function that should inhibit tumor formation and that could also be involved in spontaneous tumor regression [9]. Spontaneous tumor regression does occur in neuroblastoma, at a higher frequency than in any other cancer type. This process resembles the physiological concurrence of massive cellular suicide (apoptosis) and differentiation of a few neurons along the sympathoadrenal cell lineage in the normal development of the sympathetic nervous system. Spontaneous regression is most frequently observed in a subset of disseminated MYCN single-copy neuroblastomas (non-amplified (NA)), termed stage 4s (stage 4s-NA) [10]. However, population-based screening studies for neuroblastomas in Japan, Quebec and Germany suggest that spontaneous regression also occurs in other neuroblastoma subtypes, predominantly localized (stages 1, 2, 3) neuroblastomas (localized-NA) [11-13]. Paradoxically, MYCN mRNA and protein levels are higher in favorable localized-NA and, particularly, in stage 4s-NA tumors than in stage 4-NA tumors with poor outcome [14-16], but they do not reach the levels observed in MYCN amplified tumors. In line with this, neuroblastoma cells with elevated MYCN expression retain their capacity to undergo apoptosis [17] or neuronal differentiation [18]. Thus, it has been speculated that MYCN does not only mediate malignant progression in MYCN amplified tumors, but is also either involved or at least compatible with spontaneous regression in favorable neuroblastomas. In contrast, a functional role of MYCN in stage 4-NA tumors with low MYCN levels is questionable. Here, other transcription factors or pathways within or outside the MYC family of transcription factors could be more relevant. Neuroblastoma-derived cell lines that lack amplified MYCN generally express c-MYC rather than MYCN, often at higher levels than normal tissues [19,20]. However, transcriptional activity of MYCN or c-MYC as reflected by the transcript levels of direct MYCN/c-MYC target genes in relation to MYCN and c-MYC levels has not yet been defined in neuroblastoma subtypes. Here, we defined a core set of MYCN and c-MYC target genes by using oligonucleotide microarrays and a neuroblastoma cell line that allows conditional expression of MYCN or c-MYC. Direct regulation of these target genes by MYCN/c-MYC was assessed by analyzing the binding of MYCN and c-MYC protein to target gene promoters using PCR- and array-based chromatin immunoprecipitation (ChIP and ChIP-chip, respectively) in different neuroblastoma cell lines. We further investigated the expression of these direct MYCN/c-MYC target genes in relation to MYCN and c-MYC expression in different clinical neuroblastoma subtypes. In addition, the association of MYCN/c-MYC target gene expression with overall survival independent of the well-established markers - amplified MYCN, disease stage and age at diagnosis - was demonstrated. Results Inverse correlation of MYCN and c-MYC expression in neuroblastoma subtypes c-MYC mRNA levels are very low in MYCN amplified tumors (Figure 1), which is due to high MYCN protein repressing c-MYC mRNA expression [20]. Previous quantitative PCR analyses in a cohort of 117 neuroblastoma patients revealed that mRNA levels of MYCN are significantly lower in stage 4-NA than in stage 4s-NA (p = 0.008) and localized-NA neuroblastomas (stages 1, 2, 3; p = 0.03) [14]. To test whether this lower expression of MYCN in stage 4-NA tumors is due to elevated c-MYC activity that represses MYCN expression, we analyzed c-MYC and MYCN mRNA levels in a cohort of 251 primary neuroblastoma tumors using a customized 11K oligonucleotide microarray (other MYC gene family members were not differently expressed (data not shown)). Although c-MYC mRNA levels were not significantly higher in stage 4-NA (n = 52) than in localized-NA tumors (n = 138), we found an inverse correlation of MYCN and c-MYC expression between stage 4s-NA (n = 30) and stage 4-NA tumors. Stage 4-NA tumors showed lower expression of MYCN and higher expression of c-MYC, whereas stage 4s-NA tumors showed lower expression of c-MYC and higher expression of MYCN (Figure 1; p = 0.008 for c-MYC, p = 0.07 for MYCN). Figure 1 Inverse correlation of MYCN and c-MYC mRNA levels in neuroblastoma subtypes. Relative mRNA expression is shown for MYCN and c-MYC as well as for MDM2, DKC1, and PTMA, three direct targets of MYCN/c-MYC. Data are represented as box plots: horizontal boundaries of boxes represent the 25th and 75th percentile. The 50th percentile (median) is denoted by a horizontal line in the box and whiskers above and below extend to the most extreme data point, which is no more than 1.5 times the interquartile range from the box. A set of 251 primary neuroblastoma tumors was analyzed consisting of 138 localized-NA (stage 1/2/3), 30 stage 4s-NA, 52 stage 4-NA and 31 MYCN amplified (AMP) neuroblastoma tumors. Gene expression levels from stage 4s-NA, stage 4-NA, and MYCN amplified tumors were compared pair-wise with those of localized-NA tumors as reference. Differential gene expression was assessed for each gene by using the Mann-Whitney test (cut-off of p 4-fold enrichment for one and >2-fold enrichment of at least one of the two neighboring probes. MYCN/c-MYC-binding is color-coded as follows: blue, c-MYC binding; red, MYCN/c-MYC binding; dark red, MYCN binding; light yellow, lack of MYCN/c-MYC binding. Hierarchical clustering was used to group neuroblastoma cell lines according to their MYCN/c-MYC-binding pattern. Differentiation between MYCN and c-MYC-binding was mainly achieved through the monoclonal MYCN antibody. The polyclonal antibody against c-MYC also gave positive binding signals for a large set of analyzed target gene promoters in neuroblastoma cell lines with high MYCN that lack c-MYC expression (SH-EP MYCN , IMR5/75 and Kelly). We used SH-EP MYCN cells for a global search of MYCN and c-MYC target genes in neuroblastoma cells using a customized neuroblastoma oligonucleotide microarray (11K, Agilent) that was enriched with probes for genes differentially expressed in neuroblastoma subtypes and for direct MYCN/c-MYC target genes [14,24]. Gene expression profiles of SH-EP MYCN cells at 2, 4, 8, 12, 24, and 48 hours after targeted MYCN expression were generated. Self-organizing maps (SOMs) were used to capture the predominant pattern of gene expression. This analysis yielded 504 clusters (best matching units (BMUs)) consisting, on average, of 20 clones per cluster (Additional data file 1). We searched for clusters with characteristic gene expression profiles of direct MYCN/c-MYC target genes. In addition, known c-MYC target genes from a public database [25] and known MYCN target genes from a literature search were mapped to the 504 clusters (Additional data file 2). A significant enrichment of known MYCN/c-MYC targets was found in 6 clusters (clusters 140, 168, 195, 280, 308, and 336; p 4-fold enrichment for one probe together with >2-fold enrichment for at least one of the two neighboring probes compared to input control. In addition, we manually inspected each of the MYCN and c-MYC-binding profiles from the 147 genes. Seven genes were excluded from the analysis because the probe sets for the genes mapped within the genes but outside the target gene promoter regions (all profiles for Kelly and SJ-NB12 cell lines are given in Additional data files 3 and 4, respectively; MYCN- and c-MYC-binding results are given in Additional data files 5-7). We also performed PCR-based ChIP for selected candidate genes (n = 13; Additional data file 8), which all showed analogous results to ChIP-chip (data not shown). Almost all 140 target gene promoters showed binding of MYCN and/or c-MYC in the six analyzed neuroblastoma cell lines as measured by ChIP-chip (Figure 2b). Intriguingly, hierarchical clustering of neuroblastoma cell lines according to the MYCN/c-MYC-binding pattern clearly separated MYCN- and c-MYC-expressing neuroblastoma cell lines. Differentiation between MYCN and c-MYC binding was mainly achieved through the monoclonal anti-MYCN antibody. The polyclonal antibody against c-MYC also gave positive binding signals for a large set of target gene promoters in neuroblastoma cell lines with high MYCN that lack detectable c-MYC expression (SH-EP MYCN , IMR5/75 and Kelly). This was most likely due to unspecific binding of the polyclonal c-MYC antibody to MYCN in these cells. Nevertheless, the lack of binding of MYCN to a large set of target gene promoters in the c-MYC-expressing cells, SJ-NB12 and SY5Y, and the positive binding of c-MYC to almost all of these target gene promoters in these cells allowed the distinction between MYCN and c-MYC. Taken together, these results indicate that the genes from subgroups I and II represent a core set of target genes directly regulated by either MYCN or c-MYC in neuroblastoma cells dependent on which MYC protein is expressed. Gradual increase of MYCN/c-MYC target gene expression from stage 4s-NA through stage 4-NA to MYCN amplified tumors To determine transcriptional activity of MYCN/c-MYC proteins in primary neuroblastomas (n = 251), we analyzed differential expression of subgroup I and II genes in neuroblastoma subtypes using the Global test as proposed by Goeman et al. [27]. Almost all these genes (154 of 167; 92%) showed highest expression in MYCN amplified tumors, suggesting that regulation of these genes by MYCN is similar in neuroblastoma cell lines and tumors. Compared to localized-NA tumors (stages 1, 2, 3), expression of subgroup I and II genes was significantly associated with stage 4s-NA (p = 0.002), stage 4-NA (p < 0.001) and MYCN amplified tumors (p < 0.001). Global test results further indicated that an increasing number of MYCN/c-MYC target genes was induced from stage 4s-NA through stage 4-NA to MYCN amplified tumors (Additional data files 9-11). To further illustrate this, we grouped each of the 154 genes into one of four classes based on pair-wise comparisons (Mann-Whitney test, cut-off p < 0.05). These were, compared to localized-NA tumors: overexpressed in MYCN amplified and in stage 4s-NA tumors (class 1); overexpressed in MYCN amplified, stage 4-NA and stage 4s-NA tumors (class 2); overexpressed in MYCN amplified tumors (class 3); overexpressed in MYCN amplified and stage 4-NA tumors (class 4) (Figure 3). Compared to localized-NA tumors, 25 (16%) of the 154 MYCN/c-MYC target genes, including CCT4, FBL, MDM2, NCL, NPM1, PTMA, and TP53, were expressed at higher levels in stage 4s-NA tumors (Table 1). Eighty-eight (57%) of the 154 MYCN/c-MYC target genes, including 21 of those overexpressed also in stage 4s-NA tumors, were expressed at higher levels in stage 4-NA than in localized-NA tumors (Table 1, class 2; Additional data file 5). Accordingly, stage 4-NA tumors shared overexpression of 68 of 154 direct MYCN/c-MYC target genes (44%), including AHCY, RUVBL1, PHB, CDK4, and MRPL3, with MYCN amplified tumors. Together, this indicates that besides MYCN amplified tumors, stage 4-NA tumors, and to a lesser extent stage 4s-NA tumors, also show higher MYCN/c-MYC activity compared to localized-NA tumors. In line with this, we also found lower mRNA levels of an increasing number of MYCN/c-MYC repressed genes from stage 4s-NA (10 out of 68 (15%) in vitro validated repressed genes that are also lower in MYCN amplified tumors) through stage 4-NA (34 out of 68 (50%)) to MYCN amplified tumors (68 out of 102 in vitro validated repressed genes had the lowest expression levels in MYCN amplified tumors (67%)). Based on the relative expression of MYCN and c-MYC in neuroblastoma subtypes, we propose that elevated MYCN activity in stage 4s-NA tumors induces only a restricted set of MYCN/c-MYC target genes, whereas elevated c-MYC activity in stage 4-NA tumors induces a larger set of MYCN/c-MYC target genes. Table 1 MYCN/c-MYC target genes overexpressed in stage 4s-NA compared to localized-NA tumors (classes 1 and 2) Probe Gene name Class BMU Group MYCN/c-MYC-fold change* c-MYC target DB† Validated by ChIP‡ A_24_P311604 C4orf28 1 195 I 1.38 + A_23_P102420 CCT4 1 168 I 1.31 + A_23_P5551 NCL 1 308 II 1.69 Up + A_23_P44836 NT5DC2 1 140 I 1.40 + A_32_P139196 C13ORF25V_1 2 308 II 3.83 ND A_24_P133488 CDCA4 2 140 I 1.45 + A_23_P137143 DKC1 2 308 II 1.93 Up + A_23_P216396 EXOSC2 2 308 II 1.83 + A_23_P78892 FBL 2 195 I 1.93 Up + A_24_P228796 GAGE7B 2 195 I 1.27 ND A_23_P41025 GNL3 2 308 II 1.80 Up ND A_32_P8120 GNL3 2 308 II 1.81 Up ND A_23_P398460 HK2 2 280 II 1.71 Up + Hs172673.9 Hs172673.9 2 168 I 1.73 + A_23_P502750 MDM2 2 336 II 1.19 ChIP + A_23_P92261 MGC2408 2 280 II 2.14 + A_23_P50897 MKI67IP 2 280 II 1.97 Up + A_23_P214037 NPM1 2 140 I 1.61 Up + A_23_P57709 PCOLCE2 2 308 II 2.40 + A_24_P34632 PTMA 2 308 II 2.21 Up + A_23_P126825 SLC16A1 2 195 I 1.22 + A_23_P126291 SNRPE 2 336 II 1.49 + A_23_P117068 SNRPF 2 336 II 1.44 + A_23_P31536 SSBP1 2 336 II 1.24 + A_23_P26810 TP53 2 140 I 1.44 Up + *Fold change expression in SH-EP MYCN cells after MYCN induction. †c-MYC target gene database entry [25]: Up, upregulated; ChIP, validated by ChIP. ‡Validation of MYCN/c-MYC binding using ChIP in this study (Additional data files 5-7). BMU, best matching unit; ND, not determined. Figure 3 Expression of MYCN/c-MYC target genes in neuroblastoma subtypes. Differential expression was analyzed for each of the genes (n = 154) in MYCN amplified (AMP), stage 4s-NA and stage 4-NA tumors using localized-NA (stage 1/2/3) tumors as reference in pair-wise comparisons (Mann-Whitney test, cut-off p < 0.05, black). We grouped each of these 154 genes into one of four classes based on their relative expression in clinically relevant neuroblastoma subtypes. These classes were, compared to localized-NA tumors: overexpressed in MYCN amplified and in stage 4s-NA tumors (class 1; CCT4 and NCL); overexpressed in MYCN amplified, stage 4-NA and stage 4s-NA tumors (class 2; TP53 and FBL); overexpressed in MYCN amplified tumors (class 3; MTHFD2 and EEF1E1); and overexpressed in MYCN amplified and stage 4-NA tumors (class 4; AHCY and MRPL3). High expression of MYCN/c-MYC target genes is a robust marker of poor overall survival independent of genomic MYCN status, age at diagnosis and disease stage Having shown that MYCN/c-MYC target gene activation is also associated with distinct neuroblastoma subtypes, we wanted to test whether MYCN/c-MYC activity as determined by the expression levels of their target genes is associated with overall survival and improves outcome prediction independent of known risk markers. We used the Global test to test the influence of each of the 504 experimentally defined gene clusters on overall survival directly, without the intermediary of single gene testing. The p-values for each cluster were adjusted for multiple testing and ranked according to their association with overall survival. Table 2 gives the association with overall survival of the six MYCN/c-MYC target gene clusters and the rank in relation to all other clusters. In a separate analysis, we determined the association with overall survival for each of the 504 experimental gene clusters adjusted for amplified MYCN, stage 4 versus stages 1, 2, 3, and 4s, and age at diagnosis ≥1.5 years (Table 2). These well-established risk markers highly correlated with poor outcome in univariate analyses (p < 0.001 for each of these three markers). As expected, the Global test without adjustment for co-variables indicated that all MYCN/c-MYC target gene clusters were significantly associated with poor overall survival (p < 0.001). Intriguingly, all six MYCN/c-MYC target gene clusters remained significantly associated with overall survival after adjusting for amplified MYCN, stage 4 versus stages 1, 2, 3, and 4s, and age at diagnosis ≥1.5 years. Of note, two of the MYCN/c-MYC target gene clusters (clusters 168 and 140, both from subgroup I showing a higher responsiveness to c-MYC than to MYCN in SH-EP MYCN ) revealed the strongest association with overall survival of all 504 clusters after adjusting for co-variables (Table 2). Figure 4 shows the association with overall survival for each gene from cluster 168 with and without adjustment for co-variables. Most of the genes within this cluster, such as AHCY, ARD1A, CDK4, HSPD1, PHB, RUVBL1, and TRAP1, remained associated with overall survival after adjustment for co-variables. A less significant association with overall survival was observed for clusters with MYCN/c-MYC repressed genes: clusters 454, 482, 484, and 486 were associated with poor overall survival without adjustment for co-variables in the Global test (p < 0.001, adjusted for multiple testing), but they showed no significant association with poor overall survival when adjusting for the co-variables amplified MYCN, stage 4 versus stages 1, 2, 3, and 4s, and age at diagnosis ≥1.5 years. We also asked whether direct MYCN/c-MYC target genes as defined by our analyses are represented in previously published gene expression-based classifiers that distinguish low-risk from high-risk neuroblastomas independent of other risk markers. Gene lists from these studies hardly overlapped, making interpretation difficult. The overlap with our MYCN/c-MYC target gene list was defined by using the gene names as common identifiers. Indeed, different genes defined by our study as direct MYCN/c-MYC target genes were represented in the gene expression classifier gene lists: from the 44 genes overexpressed in high-risk neuroblastomas independent of other markers described by Schramm et al. [28], we identified 10 genes directly regulated by MYCN/c-MYC (DDX21, SCL25A3, EIFA4A2, NME1, NME2, TKT, LDHA, LDHB, HSPD1, HSPCB); from the 20 genes overexpressed in high-risk neuroblastomas independent of other markers described by Ohira et al. [29], we identified 5 genes directly regulated by MYCN/c-MYC (EEF1G, AHCY, TP53, ENO1, TKT); and from the 66 genes overexpressed in high-risk neuroblastomas independent of other markers described by Oberthuer et al. [24], we identified 7 genes directly regulated by MYCN/c-MYC (PRDX4, MRPL3, SNRPE, FBL, LOC200916, PAICS, AHCY; Figure 5). Together, these results show that MYCN/c-MYC activity as determined by the expression status of a subset of MYCN/c-MYC target genes is significantly associated with poor overall survival independent of other established markers and is a consistent element of gene expression-based neuroblastoma risk classification systems. Table 2 Association of MYCN/c-MYC target gene clusters with overall survival in primary neuroblastomas (n = 251) Cluster Number of genes Rank OS* p-value OS† Rank OS with CV* p-value OS with CV† 168 (I) 19 3 <0.0001 1 0.0004 140 (I) 38 4 <0.0001 2 0.0006 195 (I) 21 31 <0.0001 12 0.0060 308 (II) 33 18 <0.0001 26 0.0161 280 (II) 32 29 <0.0001 37 0.0232 336 (II) 26 51 <0.0001 45 0.0280 *Rank of all 504 clusters tested for association with overall survival (OS) using the Global test without and with adjustment for co-variables (CV; amplified MYCN, stages 1, 2, 3, 4s versus 4, age at diagnosis ≥1.5 years). † p-value from Global test adjusted for multiple testing. In the Cluster column, I or II gives the cluster group as defined by the SOM analysis using SH-EP MYCN cells. Figure 4 Association of cluster 168 genes with overall survival. The two gene plots illustrate the influence on overall survival of each gene from cluster 168. The gene plot gives the influence on overall survival without (left) and with (right) adjustment for the variables genomic MYCN status, age at diagnosis (≥1.5 years), and disease stage (stages 1, 2, 3, 4s versus stage 4). The gene plot shows a bar and a reference line for each gene tested. In a survival model, the expected height is zero under the null hypothesis that the gene is not associated with the clinical outcome (= reference line). Marks in the bars indicate by how many standard deviations the bar exceeds the reference line. The bars are colored to indicate a negative (red) association of a gene's expression with overall survival. In addition, the boxplot class is given for each gene. Figure 5 Representation of MYCN/c-MYC target genes in a gene expression-based neuroblastoma risk stratification system. Two-way hierarchical cluster analysis using 144 oligonucleotide probes from the gene expression-based classifier and the 251 patients from the entire cohort. Clinical characteristics (outcome, white = no event, gray = relapse/progression, black = death due to neuroblastoma; genomic MYCN status, white = NA, black = amplified; chromosome 1p status, white = normal, black = 1p deleted, gray = not available; chromosome 11q status, white = normal, black = 11q deleted, gray = not available; age at diagnosis, white <1.5 years, black ≥1.5 years; disease stage, white = stage 1, 2, gray = stage 3, yellow = stage 4s, black = stage 4) are added to the heatmap of gene expression. The gene expression cluster with direct MYCN/c-MYC target genes is highlighted. The Rank Classifier column gives the classifier rank found by the Prediction Analysis for Microarrays algorithm and a complete 10-times-repeated 10-fold cross validation. The Cluster column gives the results from the SOM analysis using gene expression profiles from SH-EP MYCN cells. The MYCN/c-MYC regulated column gives the fold changes after MYCN induction. The Ebox column gives the position of a canonical E-box in the promoter. The c-MYC TGDB column gives the entries in the public c-MYC target gene database. *UP, upregulated. Discussion In this study, we analyzed MYCN and c-MYC activity as reflected by the expression levels of a core set of direct MYCN/c-MYC targets in neuroblastoma subtypes. As expected, the highest expression levels of MYCN/c-MYC targets were observed in MYCN amplified tumors. However, we found that besides MYCN amplified tumors, subtypes of MYCN single-copy tumors, namely stage 4-NA and, to a lesser extent, stage 4s-NA, also showed increased MYCN/c-MYC target gene activation compared to localized-NA tumors. In general, low MYCN mRNA and protein levels are found in most stage 4-NA tumors [14-16], which does not explain the high mRNA levels of MYCN/c-MYC target genes in this subtype. Here, we describe an inverse correlation of MYCN and c-MYC expression levels in stage 4-NA and stage 4s-NA tumors. From experiments in neuroblastoma cell lines, it is known that MYCN and c-MYC control their expression via autoregulatory loops and via repressing each other at defined promoter sites [20]. Neuroblastoma cell lines with high expression of MYCN as a result of amplification lack c-MYC expression. Whenever MYCN and c-MYC are co-expressed in neuroblastoma cell lines, c-MYC expression predominates. Together, this suggests that increased activity of c-MYC represses MYCN in a substantial number of stage 4-NA tumors. In contrast, an inverse regulation, namely the repression of c-MYC by MYCN, is found in MYCN amplified and, to a lesser extent, in stage 4s-NA tumors. It is important to note that localized-NA tumors also express MYCN as well as c-MYC and it is likely that they are active because these tumors frequently show high tumor cell proliferation indices [14]. Nevertheless, in localized-NA tumors, we did not observe that one MYC transcription factor dominates over the other, such as in the other neuroblastoma subtypes. Our findings further indicate that MYCN/c-MYC target gene activation gradually increases from stage 4s-NA through stage 4-NA to MYCN amplified tumors. High expression of a large number of MYCN/c-MYC target genes was found in stage 4-NA and MYCN amplified tumors, but not in stage 4s-NA tumors, which is probably involved in the divergent clinical outcome of these subtypes. This also suggests that MYCN in stage 4s tumors is a weaker transactivator than c-MYC in stage 4-NA tumors. Whether this effect is due to the cellular context in which they are expressed and/or due to different functions of the two MYC proteins in neuroblastoma cells is unclear. In favor of a cellular context factor, we observed that promoter constructs from the PTMA gene, which is highly expressed in stage 4s NA and MYCN amplified tumors, showed a strong activation in N-type but not S-type neuroblastoma cell lines despite similar MYCN protein levels (unpublished data). In favor of different functions of the two MYC proteins, our analyses in SH-EP MYCN cells suggest that a large number of MYCN/c-MYC target genes (subgroup I genes) are less responsive to MYCN than to c-MYC. Another unsolved question is which molecular mechanisms induce elevated MYCN activity in stage 4s-NA tumors or elevated c-MYC activity in stage 4-NA tumors. Candidate pathways involved in differential regulation of MYC proteins are the Sonic hedgehog pathway (Shh) for MYCN activation [30] and the Wnt/beta-catenin pathway for c-MYC activation [31,32]. However, we observed that c-MYC mRNA levels are not significantly higher in stage 4-NA than in localized-NA tumors. This suggests that molecular mechanisms that increase c-MYC protein abundance/stability or simply c-MYC activity are involved in MYCN/c-MYC target gene activation in stage 4 tumors. Our data are in line with a model where stage 4s-NA tumors exhibit a moderate MYCN function gain compared to localized-NA tumors. Both subtypes usually have favorable outcome. Most localized-NA tumors are cured by surgery alone or even regress spontaneously. Stage 4s-NA tumors frequently regress spontaneously but regression can also be induced by a 'mild' chemotherapy. We found that stage 4s-NA tumors express, on average, the highest MYCN mRNA levels of all non-amplified tumors [14]. From the experimentally defined direct MYCN target genes, only a restricted set of 25 genes, including CCT4, FBL, MDM2, NCL, NPM1, PTMA, and TP53, was overexpressed in stage 4s-NA compared to localized-NA tumors, indicating that elevated MYCN in stage 4s-NA tumors only partially activates its downstream target genes. On the one hand, this suggests that moderate MYCN function gain in stage 4s-NA tumors is involved in the metastatic phenotype. On the other hand, moderate MYCN function gain in this subtype is still compatible with, or might even favor, spontaneous regression. From the list of MYCN target genes overexpressed in stage 4s-NA tumors, TP53 as a pro-apoptotic gene, and MDM2, coding for the direct inhibitor of p53 and mediating pro-tumorigenic activities, are strong candidates to be involved in the unique phenotype of stage 4s-NA tumors. However, it is important to note that TP53 and MDM2 are co-expressed at higher levels also in stage 4-NA and MYCN amplified tumors. Both subtypes initially respond to therapy, but rapidly acquire resistance and frequently show progression/relapse, suggesting that additional conditions activating MDM2 and/or suppressing TP53 functions are acquired. In line with this, alterations disrupting the p14-MDM2-p53 pathway, such as MDM2 amplification, p14 methylation/deletion, and TP53 mutations are found in neuroblastoma cell lines that were established from relapsed patients [33]. In this context, it remains to be shown whether small compounds that selectively inhibit MDM2, such as nutlin-3, and that induce proliferation arrest and apoptosis in neuroblastoma cell lines [34,35] represent a new therapeutic option for high-risk neuroblastomas. Conclusions High expression of a defined subset of direct MYCN/c-MYC target genes turned out to be a robust marker for poor overall survival independent of the established markers, amplified MYCN, disease stage (stage 4 versus stages 1, 2, 3, and 4s) and age at diagnosis (≥1.5 years). Recently, several gene expression-based neuroblastoma risk stratification systems have been developed that predict outcome more accurately than established risk markers [24,28,29]. Unfortunately, the classifier gene lists emerging from these studies hardly overlap, which has been ascribed to the different composition of the investigated cohorts and the different high-throughput gene expression platforms used. Our data show that markers of increased MYCN/c-MYC activity are consistently represented in these classifier gene lists, indicating that a gene expression-based classifier that reflects MYCN/c-MYC function should make an attractive tool for neuroblastoma classification and risk prediction. Materials and methods Patients All patients from this study (n = 251) were enrolled in the German Neuroblastoma Trials NB90-NB2004 with informed consent and diagnosed between 1989 and 2004 (patient characteristics are in Additional data files 2 and 12). Tumor samples were collected prior to any cytoreductive treatment. The only criterion for patient selection was availability of sufficient amounts of tumor material. Tumor specimens were checked for at least 60% tumor content. Neuroblastoma sample preparation and gene expression analysis Gene expression profiles from the tumors were generated as dye-flipped dual-color replicates using customized 11K oligonucleotide microarrays as previously described [24]. The 11K Agilent microarray was constructed in our laboratory based on extensive neuroblastoma transcriptome information from different whole-genome analyses from primary tumors and neuroblastoma cell lines. These also include comparative transcriptome analysis of MYCN amplified versus not amplified tumors as well as of neuroblastoma cell lines with variable/conditional MYCN/c-MYC expression that allowed the enrichment with MYCN/c-MYC-regulated genes [14,24] (unpublished data). The reference for each tumor RNA was an RNA pool of 100 neuroblastoma tumor samples. Data normalization and quality control is described in Additional data file 2. All raw and normalized microarray data are available at the ArrayExpress database (Accession: E-TABM-38) [36]. Neuroblastoma cell line experiments and SOM analysis The SH-EP MYCN cell line, previously also denoted as TET21N [23], expressing a MYCN transgene under the control of a tetracycline-repressible element was used to generate gene expression profiles from different time points after MYCN induction showing variable MYCN and c-MYC levels. RNA isolation from SH-EP MYCN cells was performed as previously described [14]. Gene expression profiles were generated as dye-flipped dual-color replicates using the same customized 11K oligonucleotide microarray platform used for the tumor samples. The reference for RNA from SH-EP MYCN cells after MYCN induction was RNA from SH-EP MYCN cells cultured in parallel that lack MYCN expression. Gene expression profiles from SH-EP MYCN cells with variable MYCN and c-MYC levels were taken for a SOM analysis (Additional data file 2). Protein expression was assessed by immunoblotting using 50 μg of total cell lysates from the cell line experiments as previously described [37]. Blots were probed with antibodies directed against MYCN (SantaCruz, sc-53993, Santa Cruz, CA, USA) and c-MYC (SantaCruz, sc-764, Santa Cruz, CA, USA). ChIP, ChIP-chip and protein analysis Chromatin immunoprecipitation was performed as described previously [38,39] using 10 μg of MYCN (SantaCruz, sc-53993), c-MYC (SantaCruz, sc-764) [40,41] and normal mouse IgG (SantaCruz, sc-2025) antibodies and Dynabeads ProteinG (Invitrogen, Carlsbad, CA, USA). Eluted and purified MYCN-ChIP-DNA (1 μl) of IMR5/75 and SH-EP MYCN was used as a template in PCR reactions running for 35 cycles. The primer sequences are given in Additional data file 8. In addition, ChIP-DNA templates from SH-EP MYCN , SH-EP, Kelly, IMR5/75, SJNB-12 and SY5Y cells using MYCN and c-MYC antibodies were amplified for DNA microarray analysis (Agilent Human Promoter ChIP-chip Set 244K) using the WGA (Sigma-Aldrich, St. Louis, MO, USA) method [42]. DNA labeling, array hybridization and measurement were performed according to Agilent mammalian ChIP-chip protocols. For the visualization of ChIP-chip results, the cureos package v0.2 for R was used (available upon request). The in silico promoter analysis for the identification of putative MYC binding sites (canonical and non-canonical E-boxes) is described in Additional data file 2. Differential gene expression and survival analysis Differential gene expression of MYCN/c-MYC and their target genes in neuroblastoma tumors was evaluated for stage 4s-NA, stage 4-NA and MYCN amplified using localized-NA tumors (stages 1, 2, 3) as reference using Goeman's Global test and the Wilcoxon rank sum test. A result was judged as 'statistically significant' at a p-value of 0.05 or smaller. Differential expression of MYCN was evaluated in two partially overlapping cohorts, one measured by quantitative PCR [14] and the other by oligo microarray (the overlap was 101 patients). To test the association of MYCN in vitro clusters with overall survival (death due to neuroblastoma disease), Goeman's Global test was used [27]. To evaluate the influence of gene expression on outcome independent of established markers, the Global test was adjusted for the following co-variables: genomic MYCN status, stage of the disease (stage 4 versus stages 1, 2, 3, and 4s), and age at diagnosis (≥1.5 years versus <1.5 years). Because of multiple testing of probably dependent gene clusters, p-values were adjusted according to Benjamini and Yekutieli [43] to control the false discovery rate of 5%. Abbreviations ChIP, PCR-based chromatin immunoprecipitation; ChIP-chip, array-based chromatin immunoprecipitation; NA, non-amplified; SOM, self-organizing map. Authors' contributions FW designed and coordinated the study. FW and DM interpreted results and drafted the manuscript. AO, MF, AB, BB and FW carried out array-based expression profiling and data analyses of neuroblastoma tumor samples and cell lines. BH was responsible for clinical data management. TB and RK performed in silico promoter analyses. JV and FP contributed samples and performed literature searches of MYCN/c-MYC target genes. DM performed chromatin immunoprecipitation experiments. DM, TB and FW analyzed ChIP-chip data. AB, BH and FW carried out global test and survival analyses. FW, DM, KOH, JV, FP and MS contributed to the manuscript. All authors read and approved the final manuscript. Additional data files The following additional data are available. Additional data file 1 is a figure showing a Cluster map of genetic programs regulated by conditional expression of c-MYC and MYCN proteins in SH-EPMYCN cells. Additional data file 2 is a document describing in more detail the methods and materials. Additional data files 3 and 4 are sets of figures showing ChIP-chip results of MYCN/c-MYC target genes in the Kelly and SJ-NB12 cell lines. Additional data files 5, 6 and 7 are tables listing MYCN/c-MYC target genes overexpressed in stage 4s-NA, stage 4-NA and MYCN amplified tumors, respectively, compared to localized-NA tumors. Additional data file 8 is a table of genes and primers selected to confirm ChIP-chip results. Additional data files 9, 10 and 11 are figures showing the association of MYCN/c-MYC induced genes with neuroblastoma subtypes using the Global test. Additional data file 12 is a table providing patient data. Supplementary Material Additional data file 1 Cluster map of genetic programs regulated by conditional expression of c-MYC and MYCN proteins in SH-EPMYCN cells. Click here for file Additional data file 2 Detailed methods and materials. Click here for file Additional data file 3 ChIP-chip results of MYCN/c-MYC target genes in the Kelly cell line. Click here for file Additional data file 4 ChIP-chip results of MYCN/c-MYC target genes in the SJ-NB12 cell line. Click here for file Additional data file 5 MYCN/c-MYC target genes, which grouped in class 1 and 2. Click here for file Additional data file 6 MYCN/c-MYC target genes, which grouped in class 4. Click here for file Additional data file 7 MYCN/c-MYC target genes, which grouped in class 3. Click here for file Additional data file 8 Genes and primers selected to confirm ChIP-chip results. Click here for file Additional data file 9 Association of MYCN/c-MYC induced genes with stage 4s-NA neuroblastomas using the Global test. Click here for file Additional data file 10 Association of MYCN/c-MYC induced genes with stage 4-NA neuroblastomas using the Global test. Click here for file Additional data file 11 Association of MYCN/c-MYC induced genes with MYCN amplified neuroblastomas using the Global test. Click here for file Additional data file 12 Patient data. Click here for file
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                Author and article information

                Journal
                BMC Dev Biol
                BMC Developmental Biology
                BioMed Central
                1471-213X
                2011
                3 January 2011
                : 11
                : 1
                Affiliations
                [1 ]Department of Pediatrics, University Hospital of North-Norway, 9038 Tromsø, Norway
                [2 ]Department of Pediatric Research, Institute of Clinical Medicine, University of Tromsø, 9037 Tromsø, Norway
                Article
                1471-213X-11-1
                10.1186/1471-213X-11-1
                3022612
                21194500
                236b59f9-a323-47d1-a049-8588c36c3a06
                Copyright ©2011 Henriksen et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<url>http://creativecommons.org/licenses/by/2.0</url>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 5 August 2010
                : 3 January 2011
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                Methodology Article

                Developmental biology
                Developmental biology

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