A genome-wide search for promoters that respond to increased MYCN reveals both new oncogenic and tumor suppressor microRNAs associated with aggressive neuroblastoma

MYCN is a major driver of neuroblastoma tumorigenesis and MYCN amplification is the worst prognostic indicator of aggressive NB. To identify potentially therapeutic tumor suppressor microRNAs for aggressive NB, we utilized a conditional MYCN system to simulate MYCN-amplified and nonamplified tumor types and performed a genome-wide search for MYCN target microRNA promoters differentially repressed under high MYCN conditions. We identified 20 gene promoters hosting 30 microRNAs that were directly bound and differentially regulated by MYCN. Eleven of these genes showed significant clinical correlations for neuroblastoma with 4 genes linked with better survival and 7 genes linked with poor survival. Surprisingly, expression analysis of host genes and microRNAs demonstrated that 8 of 11 pairs were repressed by high levels of MYCN regardless of the clinical correlation of the host gene. We therefore predicted these intronic microRNAs would be tumor suppressors. In fact, detailed gain of function studies for two miRs, miR-591 and miR-558, confirmed potent tumor suppressive effects for miR-591 in orthotopic neuroblastoma xenografts. However, miR-558 markedly increased colony formation, proliferation, and tumor growth in vivo. Our data reveal host-gene independent functions of MYCN-target microRNAs and demonstrate that MYCN represses both tumor suppressive and proproliferative microRNAs.     

Neuroblastomas with chromosome 11q loss and single copy MYCN comprise a biologically distinct group of tumours with adverse prognosis

Neuroblastoma is a heterogeneous tumour and its effective clinical management is dependent on accurate prognostic evaluation. In approximately 25% of patients amplification of the MYCN oncogene is known to be associated with a poor outcome. In order to identify additional molecular markers with prognostic potential in non-MYCN-amplified neuroblastomas, we looked for a correlation between clinical outcome and loss of heterozygosity (LOH) on four chromosomes that frequently show alteration in neuroblastoma (chromosomes 3, 4, 11 and 14). Chromosome 11q loss (with frequent parallel loss of chromosomes 3p, 4p and/or 14q) was found exclusively in tumours without MYCN amplification and was significantly associated with poor event-free survival. The 2-year event-free survival rate for 11q LOH cases was 30%, compared to 34% for MYCN-amplified cases and 100% for cases without these abnormalities. While 11q LOH was associated predominantly with advanced-stage disease, 2 cases with low-stage disease and 11q LOH both suffered relapses. We conclude that chromosome 11q loss defines a biologically distinct group of tumours without MYCN amplification that appear to have potential for aggressive metastatic growth. Thus this genetic alteration may be an important new prognostic marker in neuroblastoma.     

Does MYCN amplification manifested as homogeneously staining regions at diagnosis predict a worse outcome in children with neuroblastoma? A Children's Oncology Group study.

PURPOSE: MYCN amplification in neuroblastoma tumor cells is manifested primarily as double minutes (dmins), whereas in cell lines it often appears in the form of homogeneously staining regions (HSR), suggesting that HSRs are associated with a more aggressive tumor phenotype and worse clinical outcome. The aim of this study was to determine whether children with neuroblastoma in which MYCN oncogene amplification is manifested as HSRs at diagnosis have a worse prognosis than those whose tumors exhibit dmins. EXPERIMENTAL DESIGN: A retrospective analysis of primary neuroblastomas analyzed for MYCN amplification by the Children's Oncology Group between 1993 and 2004 was done. Tumors with MYCN amplification were defined as having dmins, HSRs, or both (dmins + HSRs), and associations with currently used risk group stratification variables and patient outcome were assessed. RESULTS: Of the 4,102 tumor samples analyzed, 800 (19.5%) had MYCN amplification. Among the 677 tumors for which the pattern of amplification was known, 629 (92.9%) had dmins, 40 (5.9%) had HSRs, and 8 (0.1%) had dmins + HSRs. Although MYCN amplification is associated with older age, higher stage, and unfavorable histology, whether the amplification occurred as dmins or HSRs did not significantly affect these risk factors. There were no differences in the event-free survival (EFS) or overall survival in patients with MYCN amplification manifested as either dmins or HSRs (5-year EFS, 35 +/- 3% versus 38 +/- 15%; P = 0.59). Although the eight patients with dmins + HSRs fared worse than either of the individual subgroups (EFS, 18 +/- 16% versus 35 +/- 3% for dmins and 38 +/- 15% for HSRs), these differences were not significant. CONCLUSIONS: MYCN amplification in any form (HSRs or dmins) is associated with a poor outcome.     

Activation of Akt predicts poor outcome in neuroblastoma

Whereas aberrant activation of the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway, a key survival cascade, has previously been linked to poor prognosis in several human malignancies, its prognostic effect in neuroblastoma has not yet been explored. We therefore investigated the phosphorylation status of Akt, S6 ribosomal protein as target of mammalian target of rapamycin, and extracellular signal-regulated kinase (ERK) in 116 primary neuroblastoma samples by tissue microarray and its correlation with established prognostic markers and survival outcome. Here, we provide for the first time evidence that phosphorylation of Akt at serine 473 (S473) and/or threonine 308 (T308), S6 ribosomal protein, and ERK frequently occurs in primary neuroblastoma. Importantly, we identified Akt activation as a novel prognostic indicator of decreased event-free or overall survival in neuroblastoma, whereas phosphorylation of S6 ribosomal protein or ERK had no prognostic effect. In addition, Akt activation correlated with variables of aggressive disease, including MYCN amplification, 1p36 aberrations, advanced disease stage, age at diagnosis, and unfavorable histology. Monitoring Akt at T308 or both phosphorylation sites improved the prognostic significance of Akt activation in neuroblastoma specimens compared with S473 phosphorylation. Parallel experiments in neuroblastoma cell lines revealed that activation of Akt by insulin-like growth factor (IGF)-I significantly inhibited tumor necrosis factor-related apoptosis-inducing ligand- or chemotherapy-induced apoptosis in a PI3K-dependent manner because the PI3K inhibitor LY294002 completely reversed the IGF-I-mediated protection of neuroblastoma cells from apoptosis. By showing that activation of Akt correlates with poor prognosis in primary neuroblastoma in vivo and with apoptosis resistance in vitro, our findings indicate that Akt presents a clinically relevant target in neuroblastoma that warrants further investigation.     

Whole chromosome alterations predict survival in high-risk neuroblastoma without MYCN amplification

PURPOSE: Patients with stage IV neuroblastoma over the age of 500 days without MYCN amplification have a survival rate of <30% and there are currently no reliable means of predicting which of these patients will survive or succumb to the disease. The goal of this study is to develop a DNA copy number-based prognostic profile for these patients. EXPERIMENTAL DESIGN: We have used comparative genomic hybridization to identify genome copy number changes that can predict outcome in patients with stage IV neuroblastoma without MYCN amplification. RESULTS: A strong correlation of patient survival with the presence of whole chromosome changes (WCC >or=2) was observed, even in the group of patients older than 500 days at time of diagnosis. This novel prognostic marker showed a significant dependence on the date of diagnosis; patients with WCC >or=2 diagnosed after 1998 had a significantly higher probability of survival compared with those diagnosed earlier. At the same time, no such time dependence was found among the samples with WCC <2, suggesting that medical progress patients in recent years has particularly benefited those patients with a stage IV non-MYCN-amplified disease if WCC >or=2 were present. CONCLUSIONS: In this pilot study, we present a novel prognostic marker for survival of high-risk neuroblastoma patients over the age of 500 days without MYCN amplification and diagnosed after 1998. Further validation study is required to establish this risk stratification for these patients.     

Loss of heterozygosity at 1p36 independently predicts for disease progression but not decreased overall survival probability in neuroblastoma patients: a Children's Cancer Group study.

PURPOSE: To determine the independent prognostic significance of 1p36 loss of heterozygosity (LOH) in a representative group of neuroblastoma patients. PATIENTS AND METHODS: Diagnostic tumor specimens from 238 patients registered onto the most recent Children's Cancer Group phase III clinical trials were assayed for LOH with 13 microsatellite polymorphic markers spanning chromosome band 1p36. Allelic status at 1p36 was correlated with other prognostic variables and disease outcome. RESULTS: LOH at 1p36 was detected in 83 (35%) of 238 neuroblastomas. There was a correlation of 1p36 LOH with age at diagnosis greater than 1 year (P = .026), metastatic disease (P<.001), elevated serum ferritin level (P<.001), unfavorable histopathology (P<.001), and MYCN oncogene amplification (P<.001). LOH at 1p36 was associated with decreased event-free survival (EFS) and overall survival (OS) probabilities (P<.0001). For the 180 cases with single-copy MYCN, 1p36 LOH status was highly correlated with decreased EFS (P = .0002) but not OS (P = .1212). Entering 1p36 LOH into a multivariate regression model suggested a trend toward an independent association with decreased EFS (P = .0558) but not with decreased OS (P = .3687). Furthermore, allelic status at 1p36 was the only prognostic variable that was significantly associated with decreased EFS in low-risk neuroblastoma patients (P = .0148). CONCLUSION: LOH at 1p36 is independently associated with decreased EFS, but not OS, in neuroblastoma patients. Determination of 1p36 allelic status may be useful for predicting which neuroblastoma patients with otherwise favorable clinical and biologic features are more likely to have disease progression.     

Novel noncatalytic role for caspase-8 in promoting SRC-mediated adhesion and Erk signaling in neuroblastoma cells

Neuroblastomas are extremely aggressive, although heterogeneous, cancers with a poor prognosis upon metastasis. Some evidence has suggested a correlative silencing of caspase-8 with MYCN amplification in neuroblastoma. A prognostic effect of this silencing, however, has been disputed. We report here hitherto undescribed roles for caspase-8 in the modulation of cell adhesion and subsequent activation of the Erk signaling pathway. Re-expression of caspase-8 in neuroblastoma cells lacking endogenous caspase-8 expression was found to promote cell adhesion to extracellular matrix and to activate adhesion-dependent signaling pathways, such as the Erk kinase cascade. This function of caspase-8 occurred irrespective of its proteolytic activity. Additionally, a pool of caspase-8 was shown to co-localize with the Src tyrosine kinase at the cellular periphery. Furthermore, our studies showed that caspase-8 forms a physical protein complex with Src via its death effector domains (DED) and maintains the complex in a detergent-soluble fraction. We also show that the DEDs of caspase-8 alone are necessary and sufficient to recreate the adhesive and biochemical phenotypes observed with the full-length protein, suggesting that caspase-8 may exert these effects via its association with Src. This protein complex association of caspase-8 and Src, and concomitant downstream signaling events, may help reconcile why a potential tumor suppressor such as caspase-8 is rarely absent in cancers.     

GRP78 expression correlates with histologic differentiation and favorable prognosis in neuroblastic tumors

Glucose-regulated protein 78 (GRP78), an endoplasmic reticulum protein, is essential for the differentiation of neuroblastoma cells and is selectively induced when the cells are undergoing apoptosis. These findings suggest that GRP78 may affect the tumor behavior of neuroblastoma. Our study evaluates the association of clinicopathologic factors and patient survival with the expression of GRP78 in patients with neuroblastoma. GRP78 expression in 68 neuroblastic tumors was investigated semiquantitatively by immunohistochemistry. GRP78 mRNA and protein levels in 7 tumor tissues were also quantified by real-time PCR and Western blot respectively and correlated well with the immunohistochemical results. Forty (58.8%) of the 68 neuroblastic tumors showed positive GRP78 expression. The percentage of positive GRP78 immunostaining increased as the tumor histology became differentiated (p = 0.001). Furthermore, positive GRP78 expression strongly correlated with early clinical stages (P = 0.002) but inversely correlated with MYCN amplification (p = 0.001). Kaplan-Meier analysis showed that patients with positive GRP78 expression did have better survival than those with negative expression (5-year survival rate, 72.9% and 23.4% respectively, p < 0.001). Multivariate analysis further showed that GRP78 expression was an independent prognostic factor. Moreover, GRP78 expression predicted better survival in patients with either undifferentiated or differentiated histologies. GRP78 expression still had significant prognostic value when the analysis was restricted to tumors of advanced stages or without MYCN amplification. Thus, GRP78 can serve as a novel independent favorable prognostic factor for patients with neuroblastoma.     

Caspase-9 and Apaf-1 are expressed and functionally active in human neuroblastoma tumor cell lines with 1p36 LOH and amplified MYCN

Important roles have been suggested for caspase-8, caspase-9 and Apaf-1 in controlling tumor development and their sensitivity to chemotherapeutic agents. Methylation and deletion of Apaf-1 and CASP8 results in the loss of their expression in melanoma and neuroblastoma, respectively, while CASP9 localization to 1p36.1 suggests it is a good candidate tumor suppressor. The status of CASP9 and Apaf-1 expression in numerous neuroblastoma cell lines with/without amplified MYCN and chromosome 1p36 loss-of-heterozygosity (LOH) was therefore examined to test the hypothesis that one or both of these genes are tumor suppressors in neuroblastoma. Although CASP9 is included in the region encompassing 1p36 LOH in all neuroblastoma cell lines examined, the remaining CASP9 allele(s) express a functional caspase-9 enzyme. Apaf-1 is also expressed in all neuroblastoma tumor cell lines examined. Thus, the CASP9 or Apaf-1 genes do not appear to function as tumor suppressors in MYCN amplified neuroblastomas. However, approximately 20% of the neuroblastoma cell lines with methylated CASP8 alleles are also highly resistant to staurosporine (STS)- and radiation-induced cell death, presumably because cytochrome c is not released from mitochondria. This suggests that a second, smaller sub-group of MYCN amplified neuroblastoma tumors exists with defect(s) in apoptotic signaling components upstream of caspase-9 and Apaf-1. Since no consistent differences in Bcl-2, Bcl-x(L) or Bax expression were seen in the STS- and radiation-resistant neuroblastomas, it suggests that a unique mitochondrial signaling factor(s) is responsible for the defect in cytochrome c release in this sub-group of tumors.     

Calreticulin expression in neuroblastoma--a novel independent prognostic factor.

BACKGROUND: Calreticulin (CRT), an endoplasmic reticulum protein, has been reported to be essential for the differentiation of neuroblastoma (NB) cells, suggesting that CRT may affect the tumor behavior of neuroblastoma. The aim of this study was to evaluate the association of clinicopathologic factors and patient survival with the expression of CRT in patients with NB. PATIENTS AND METHODS: Sixty-eight NBs were investigated by immunohistochemical staining against CRT, and were divided into positive and negative immunostaining groups. Correlations between calreticulin expression, various clinicopathologic and biologic factors, and patient survival were studied. In seven tumor samples, CRT mRNAs and proteins were evaluated with real-time PCR and western blot, respectively, and correlated with immunohistochemical findings. RESULTS: Among 68 NBs, 32 (47.1%) showed positive CRT expression. Positive CRT immunostaining strongly correlated with differentiated histologies, as well as known favorable prognostic factors such as detected from mass screening, younger age (< or =1 year) at diagnosis and early clinical stages, but inversely correlated with MYCN amplification. Kaplan-Meier analysis revealed that NB patients with CRT expression did have better survival. Multivariate analysis demonstrated CRT expression to be an independent prognostic factor. Moreover, CRT expression also predicted better survival in patients with advanced-stage NBs, and its absence predicted poorer survival in patients whose tumor had no MYCN amplification. The amount of CRT mRNAs and proteins in NB tumor samples tested correlated well with the immunohistochemical expressions. CONCLUSIONS: CRT expression correlates with the differentiation of NB and predicts favorable survival, thereby suggesting CRT to be a useful indicator for planning treatment of NB.     

Differential patterns of microRNA expression in neuroblastoma are correlated with prognosis, differentiation, and apoptosis

Neuroblastoma accounts for 15% of pediatric cancer deaths, and although a few protein-coding genes, such as MYCN, are involved with aggressive pathogenicity, the identification of novel biological targets for therapeutic intervention is still a necessary prerequisite for improving patient survival. Expression profiling of 157 microRNA (miRNA) loci in 35 primary neuroblastoma tumors indicates that 32 loci are differentially expressed in favorable and unfavorable tumor subtypes, indicating a potential role of miRNAs in neuroblastoma pathogenesis. Many of these loci are significantly underexpressed in tumors with MYCN amplification, which have particularly poor prognoses. Interestingly, we found that miRNA expression levels substantially change in a MYCN-amplified cell line following exposure to retinoic acid, a compound which is well known for causing reductions in MYCN expression and for inducing neuroblastoma cell lines to undergo neuronal differentiation. We also show that small interfering RNA inhibition of MYCN by itself causes similar alterations in the expression of miRNA loci. In vitro functional studies of one locus, miR-184, indicate that it plays a significant role in apoptosis. The association of experimentally induced alterations of miRNA expression in neuroblastoma cell lines with differentiation or apoptosis leads us to conclude that these loci play important roles in neuroblastoma pathogenesis. We further suggest that MYCN may mediate a tumorigenic effect, in part, through directly or indirectly regulating the expression of miRNAs that are involved with neural cell differentiation and/or apoptosis, warranting substantial further studies of miRNAs as potential therapeutic targets.     

MYCN downregulates integrin alpha1 to promote invasion of human neuroblastoma cells

Neuroblastoma is a childhood tumor thought to arise through improper differentiation of neural crest cells. MYCN amplification is a prognostic factor that indicates a highly malignant disease and poor patient prognosis. Integrins are important regulators of neuroblastoma attachment and migration and participate in many aspects of metastasis. However, the role of integrins in neuroblastoma metastasis, the leading cause of death from this disease, remains less well understood. Screening of neuroblastoma cell lines for integrin mRNA expression showed that integrin alpha1 expression was higher in lines such as SK-N-SH and NB69 that do not have MYCN amplification than in cell lines such as IMR32, NB1, NB9 and NB19 that have MYCN amplification. A knockdown of MYCN in NB1 and NB19 cells resulted in increased expression of integrin alpha1, which correlated with enhanced attachment to the extracellular matrix and reduced migratory activity. In contrast, the overexpression of MYCN in SK-N-SH and NB69 cells resulted in decreased expression of integrin alpha1, which correlated with reduced attachment to the extracellular matrix and enhanced migratory activity. These results show that MYCN may limit cell adhesion to the extracellular matrix and promote cell migration by downregulating integrin alpha1.     

Loss of a FYN-regulated differentiation and growth arrest pathway in advanced stage neuroblastoma

Tumor stage, age of patient, and amplification of MYCN predict disease outcome in neuroblastoma. To gain insight into the underlying molecular pathways, we have obtained expression profiles from 94 primary neuroblastoma specimens. Advanced tumor stages show a characteristic expression profile that includes downregulation of multiple genes involved in signal transduction through Fyn and the actin cytoskeleton. High expression of Fyn and high Fyn kinase activity are restricted to low-stage tumors. In culture, expression of active Fyn kinase induces differentiation and growth arrest of neuroblastoma cells. Expression of Fyn predicts long-term survival independently of MYCN amplification. Amplification of MYCN correlates with deregulation of a distinct set of genes, many of which are target genes of Myc. Our data demonstrate a causal role for Fyn kinase in the genesis of neuroblastoma.