N-myc down regulates neural cell adhesion molecule expression in rat neuroblastoma.

In human neuroblastoma, amplification of the N-myc oncogene is correlated with increased metastatic ability. We recently showed that transfection of the rat neuroblastoma cell line B104 with an N-myc expression vector resulted in an increase in metastatic ability and a significant reduction in the expression of major histocompatibility complex class I antigens. We examined whether N-myc causes additional phenotypic changes in these cells. We showed that expression of N-myc leads to a dramatic reduction in the levels of neural cell adhesion molecule (NCAM) polypeptides and mRNAs. Spontaneous revertants of the high N-myc phenotype were found to have regained significant levels of NCAM expression, indicating that the continued expression of N-myc is required to maintain the low NCAM phenotype. NCAM was not reduced in B104 cells transfected with the neomycin resistance vector alone, and other neuronal markers were not specifically reduced in N-myc-transfected B104 cells. As NCAM functions in cell-cell adhesion, decreased NCAM expression could contribute significantly to the increased metastatic potential of N-myc-amplified neuroblastomas.     

Retinoic acid treatment of human neuroblastoma cells is associated with decreased N-myc expression

Cells from human neuroectodermal tumors (retinoblastoma and neuroblastoma) and from neuroblastoma cell lines express a gene, N-myc, which is frequently amplified in these tumors. We report here that N-myc mRNA content is markedly decreased in cells of a neuroblastoma cell line (LA-N-5) following differentiation induced with retinoic acid. Exposure of the cells to retinoic acid induced morphologic changes consistent with neuronal differentiation, and led to a 75% decrease in expression of N-myc mRNA. These results suggest that N-myc expression is intimately related to an undifferentiated phenotype in neuroblastoma cells, and support other studies which relate N-myc expression to the malignant phenotype in neuroblastoma tumors.     

Down modulation of N-myc, heat-shock protein 70, and nucleolin during the differentiation of human neuroblastoma cells.

Cultured human neuroblastoma (GOTO) cells were induced to differentiate by dibutyryl cyclic AMP (Bt2cAMP) and/or retinoic acid (RA). A combination of Bt2cAMP (1 mM) and RA (1 microM) yielded the most significant networks of neurites after 3 to 4 days, this being associated with the reduction of N-myc mRNA levels. Next, we examined several cellular genes that were possibly linked with changes in N-myc gene expression under these conditions. Among the genes examined, both nucleolin and a major heat-shock protein (hsp70) mRNAs showed changes concomitant with those in N-myc mRNA levels when induced by Bt2cAMP and RA. Dibutyryl cAMP alone induced several short cellular processes and caused a marked decrease in N-myc mRNA within 2 days. RA alone induced a few long and straight neurites along the longitudinal axis of individual cells and a significant decrease in growth rate but showed neither network formation nor a decrease in N-myc gene expression. These results indicate differential effects of Bt2cAMP and RA on the regulatory mechanisms of both cell proliferation and differentiation and also indicate a possible association of expression of N-myc gene with those of hsp70 and nucleolin genes.     

Transrepression of the N-myc expression by c-myc protein

In the human neuroblastoma cell line IMR32 the N-myc gene happens to be amplified and actively expressed, whereas no stable c-myc RNA can be detected in the same cells. In this report, we show that in IMR32 cells the expression of the N-myc gene is repressed by introduction of a c-myc expression vector (c-myc cDNA conjugated with an SR promoter). Moreover, dose response experiments showed that the amount of endogenous c-myc protein present in HeLa cells (which express c-myc but not N-myc) is enough to repress the expression of N-myc in IMR32 cells.     

Hypoxia promotes apoptosis of human neuroblastoma cell lines with enhanced N-myc expression

We have examined the effect of hypoxia and nutrient depletion on the growth of human neuroblastoma cells with normal or enhanced expression of the N-myc oncogene. The combination of both conditions reduced the growth of neuroblastoma cells with normal N-myc expression. However, this effect was much more pronounced in neuroblastoma cells with enhanced N-myc expression and eventually resulted in apoptosis, presumably by the up-regulation of CD95. Our data suggest that therapeutic induction of tumor hypoxia and nutrient depletion (for example, by anti-angiogenesis) could help to improve the outcome of patients with neuroblastomas carrying the prognostically unfavourable N-myc amplification.     

Combined RAR alpha- and RXR-specific ligands overcome N-myc-associated retinoid resistance in neuroblastoma cells

Retinoids induce human neuroblastoma cells to undergo growth inhibition and neuritic differentiation in vitro, through interactions with nuclear retinoid receptor proteins. In this study, we found that three different neuroblastoma cell lines exhibited wide variation in their responsiveness to the growth inhibitory effects of the retinoic acid receptor (RAR) agonist, all-trans-retinoic acid (aRA). Resistance to the growth inhibitory effect of aRA correlated with the presence of N-myc gene amplification and not aRA-induced RAR beta levels. Over-expression of N-myc in a neuroblastoma cell line with no endogenous N-myc expression caused a marked reduction in retinoid-induced growth inhibition. Combination of receptor-specific retinoid agonists for RXR and RAR alpha significantly enhanced the sensitivity of N-myc-amplified neuroblastoma cells to the growth inhibitory effects of aRA. Our results indicate that combination receptor-specific retinoid therapy can overcome N-myc-mediated retinoid resistance and may be a more effective chemo-preventive strategy in the disease.     

Suppression of N-myc over-expression in human neuroblastic cells by human gamma-interferon

In the previous study, it was shown that the treatment of human neuroblastoma cells with human interferon-gamma (HuIFN-gamma) induced the morphological changes. However, the treatment with human interferon-alpha (HuIFN-alpha) or -beta (HuIFN-beta) did not induce them. In the present study, the effect of HuIFNs on the overexpression of N-myc of the human neuroblastoma cells (GOTO strain) is examined. The treatment of GOTO cells with rHuIFN-gamma inhibits the overexpression of N-myc, and its degree is dependent on the duration of the treatment. However, HuIFN-alpha and HuIFN-beta did not inhibit the overexpression of N-myc. This suggests that the oncogene N-myc may have relation to the morphological differentiation of human neuroblastoma cells because only the HuIFN-gamma, which induces the morphological differentiation, inhibits the overexpression of N-myc.     

Nerve growth factor-induced differentiation of human neuroblastoma and neuroepithelioma cell lines

A series of neuroepithelioma and neuroblastoma cell lines were screened for nerve growth factor (NGF)-induced differentiation. All three neuroepithelioma cell lines and all nine neuroblastoma cell lines with amplified N-myc oncogene did not show any apparent NGF-induced differentiation. However, neurite extension was observed for three of six neuroblastoma cell lines with single-copy N-myc oncogene. The three responsive lines had a neuronal phenotype (short processes) which was enhanced by the addition of NGF. The three nonresponsive cell lines were flat without any processes. The addition of NGF to the responsive cell lines resulted in an up-regulation of neurofilament mRNA expression. Peripherin and synapsin, two markers of terminal neuronal differentiation, were not induced. There was little effect of NGF on the rate of cell growth or colony formation on soft agar. Binding of NGF to eight of the cell lines was analyzed by the method of Scatchard. Two responsive neuroblastoma cell lines and one nonresponsive neuroepithelioma cell line expressed both low- and high-affinity binding sites. Two nonresponsive neuroblastoma cell lines expressed only a small number of high-affinity binding sites, and two other nonresponsive neuroblastoma cell lines did not detectably bind NGF. Hence, NGF-induced differentiation is confined to a particular class of neural-related tumors, and, even for these cell lines, differentiation is incomplete.     

Plasma membrane redox activity correlates with N-myc expression in neuroblastoma cells.

In different neuroblastoma cell lines and transfected clones, an increasing plasma membrane redox activity correlates with amplification and enhanced expression of the N-myc oncogene. Furthermore, plasma membrane redox activity is partially inhibited by retinoic acid in neuroblastoma cells with multiple copies of the N-myc oncogene but not in neuroblastoma cells with only one copy of this gene.     

Mutually exclusive expression of a helix-loop-helix gene and N-myc in human neuroblastomas and in normal development.

We have isolated a novel human gene encoding a helix-loop-helix (HLH) protein by molecularly cloning chromosome 1p36-specific CpG islands. The gene termed heir-1 was localized to the neuroblastoma consensus deletion at 1p36.2-p36.12. Its predicted protein is 95.8% identical to the mouse HLH462 protein and has clear homology to the mouse Id and Drosophila emc proteins. Heir-1 does not encode a basic DNA binding domain as found in basic HLH proteins. The gene is expressed specifically at high abundance in adult lung, kidney and adrenal medulla, but not in adult brain. Despite prominent heir-1 expression in adrenal medulla, which is a prime target for neuroblastomas, 10 out of 12 neuroblastoma-derived cell lines revealed very low levels of heir-1 mRNA. Low heir-1 expression was generally found in tumor cell lines with N-myc overexpression, whereas the two cell lines displaying high heir-1 levels did not overexpress N-myc. Mutually exclusive expression of both genes was also found by in situ hybridization in developing mouse tissues, particularly in the forebrain neuroectoderm. We conclude that heir-1 expression is reduced specifically in the majority of neuroblastomas and suggest an inverse correlation between heir-1 and N-myc expression in neuroblastoma tumors and in embryonic development.     

N-myc amplification causes down-modulation of MHC class I antigen expression in neuroblastoma

Amplification of the N-myc gene is correlated with increased metastatic ability of human neuroblastomas. We show here that overexpression of the N-myc gene in a rat neuroblastoma cell line following gene transfer causes down-modulation of class I histocompatibility antigen expression and increases in the in vivo growth rate and metastatic ability of these cells. N-myc-mediated down-modulation of MHC class I antigen expression could be reversed by treatment with interferon without affecting the steady state level of N-myc mRNA. No effect on MHC class I antigen expression was found when the N-myc gene was expressed in rat fibroblasts, indicating that some of the effects caused by N-myc gene amplification are cell-type-specific.     

Neoplastic transformation by the human gene N-myc.

Amplification and abundant expression of a gene known as N-myc are found frequently in advanced stages of human neuroblastoma and may play a role in the genesis of several malignant human tumors. Previous studies have shown that N-myc can cooperate with a mutant allele of the proto-oncogene c-Ha-ras to transform embryonic rat cells in culture. Here we show that N-myc can also act alone to elicit neoplastic growth of an established line of rat fibroblasts (Rat-1). We used recombinant DNA vectors to express either N-myc or its kindred gene c-myc in transfected cells. Both genes caused morphological transformation, anchorage-independent growth, and tumorigenicity. We noticed two variables that appeared to influence the ability to isolate cells transformed by N-myc and c-myc: the abundance in which the genes were expressed and biological selection to eliminate untransformed cells from the cultures. Our findings sustain the belief that N-myc is an authentic proto-oncogene, lend further credibility to the role of N-myc in the genesis of human tumors, and establish a convenient assay that can be used to explore further the properties of both N-myc and c-myc.     

Akt2 Regulates Metastatic Potential in Neuroblastoma

Activation of PI3K/AKT pathway correlates with poor prognosis in patients with neuroblastoma. Our previous studies have demonstrated that PI3K/AKT signaling is critical for the oncogenic transformations induced by gastrin-releasing peptide (GRP) and its receptor, GRP-R, in neuroblastoma. Moreover, PI3K/AKT-dependent oncogenic transformations require N-myc, an extensively studied oncogene in neuroblastoma. Whether AKT directly regulates the expression of N-myc oncogene is yet to be determined. Here, we report a novel finding that of the three AKT isoforms, AKT2 specifically regulated N-myc expression in neuroblastoma cells. We also confirmed that GRP-R is upstream of AKT2 and in turn, regulated N-myc expression via AKT2 in neuroblastoma cells. Functional assays demonstrated that attenuation of AKT2 impaired cell proliferation and anchorage-independent cell growth, and decreased the secretion of angiogenic factor VEGF in vitro. Furthermore, silencing AKT2 inhibited migration and invasion of neuroblastoma cells in vitro. Xenografts established by injecting AKT2 silenced human neuroblastoma cells into murine spleen expressed decreased levels of AKT2 and resulted in fewer liver metastases compared to controls in vivo. Hence, our study highlights the potential molecular mechanism(s) mediating the oncogenic role of GRP/GRP-R and demonstrates a novel role for AKT2 in neuroblastoma tumorigenesis, indicating that targeting the GRP/GRP-R/AKT2 axis may be important for developing novel therapeutics in the treatment of clinically aggressive neuroblastoma.