N-Myc Regulates Expression of Pluripotency Genes in Neuroblastoma Including lif,klf2, klf4, and lin28b

myc genes are best known for causing tumors when overexpressed, but recent studies suggest endogenous myc regulates pluripotency and self-renewal of stem cells. For example, N-myc is associated with a number of tumors including neuroblastoma, but also plays a central role in the function of normal neural stem and precursor cells (NSC). Both c- and N-myc also enhance the production of induced pluripotent stem cells (iPSC) and are linked to neural tumor stem cells. The mechanisms by which myc regulates normal and neoplastic stem-related functions remain largely open questions. Here from a global, unbiased search for N-Myc bound genes using ChIP-chip assays in neuroblastoma, we found lif as a putative N-Myc bound gene with a number of strong N-Myc binding peaks in the promoter region enriched for E-boxes. Amongst putative N-Myc target genes in expression microarray studies in neuroblastoma we also found lif and three additional important embryonic stem cell (ESC)-related factors that are linked to production of iPSC: klf2, klf4, and lin28b. To examine the regulation of these genes by N-Myc, we measured their expression using neuroblastoma cells that contain a Tet-regulatable N-myc transgene (TET21N) as well as NSC with a nestin-cre driven N-myc knockout. N-myc levels closely correlated with the expression of all of these genes in neuroblastoma and all but lif in NSC. Direct ChIP assays also indicate that N-Myc directly binds the lif promoter. N-Myc regulates trimethylation of lysine 4 of histone H3 in the promoter of lif and possibly in the promoters of several other stem-related genes. Together these findings indicate that N-Myc regulates overlapping stem-related gene expression programs in neuroblastoma and NSC, supporting a novel model by which amplification of the N-myc gene may drive formation of neuroblastoma. They also suggest mechanisms by which Myc proteins more generally contribute to maintenance of pluripotency and self-renewal of ESC as well as to iPSC formation.     

Neural Precursor Cycling at Sonic Speed: N-Myc Pedals,GSK-3 Brakes

Signaling by the sonic hedgehog (Shh) pathway is essential for neural precursor population expansion during normal central nervous system (CNS) development, and is implicated in the childhood brain tumor, medulloblastoma. The proto-oncogene N-myc plays essential roles as a downstream effector of Shh proliferative effects in neural precursors of the cerebellum, where medulloblastomas arise. It is likely that N-Myc has analogous functions in medulloblastomas and other CNS tumors where it is highly expressed due to altered regulation or gene amplification. Myc destabilization occurs in response to phosphorylation by GSK-3β. N-Myc degradation is required for cerebellar neural precursors to exit the cell cycle. During mitosis in cerebellar neural precursors, levels of N-Myc primed for phosphorylation by GSK-3β increase, due to cdk1 complex activity towards N-Myc. GSK-3β is kept in check by insulin-like growth factor signaling, which also plays critical roles in brain development and cancer. These findings indicate that therapeutic strategies targeting N-myc and the IGF pathway might be effective against medulloblastoma.     

N-myc oncogene overexpression down-regulates leukemia inhibitory factor in neuroblastoma.

Amplification of N-myc oncogene is a frequent event in advanced stages of human neuroblastoma and correlates with poor prognosis and enhanced neovascularization. Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies, which is modulated by tumor suppressors and oncogenes. We have addressed the possibility that N-myc oncogene might regulate angiogenesis in neuroblastoma. Here, we report that experimental N-Myc overexpression results in down-regulation of leukemia inhibitory factor (LIF), a modulator of endothelial cell proliferation. Reporter assays using the LIF promoter and a series of N-Myc mutants clearly demonstrated that down-regulation of the LIF promoter was independent of Myc/Max interaction and required a contiguous N-terminal N-Myc domain. STAT3, a downstream signal transducer, was essential for LIF activity as infection with adenoviruses expressing a phosphorylation-deficient STAT3 mutant rendered endothelial cells insensitive to the antiproliferative action of LIF. LIF did not influence neuroblastoma cell proliferation suggesting that, at least in the context of neuroblastoma, LIF is involved in paracrine rather than autocrine interactions. Our data shed light on the mechanisms by which N-myc oncogene amplification enhances the malignant phenotype in neuroblastoma.