金子 伊樹

Neuroblastoma (NB) is the most common extracranial solid tumor that originates from multipotent neural crest cells. NB cell populations that express embryonic stem cell-associated genes have been identified and shown to retain a multipotent phenotype. However, whether somatic reprogramming of NB cells can produce similar stem-cell like populations is unknown. Here, we sought to reprogram NB cell lines using an integration-free Sendai virus vector system. Of four NB cell lines examined, only SH-IN cells formed induced pluripotent stem cell-like colonies (SH-IN 4F colonies) at approximately 6weeks following transduction. These SH-IN 4F colonies were alkaline phosphatase-positive. Array comparative genomic hybridization analysis indicated identical genomic aberrations in the SH-IN 4F cells as in the parental cells. SH-IN 4F cells had the ability to differentiate into the three embryonic germ layers invitro, but rather formed NBs invivo. Furthermore, SH-IN 4F cells exhibited resistance to cisplatin treatment and differentiated into endothelial-like cells expressing CD31 in the presence of vascular endothelial growth factor. These results suggest that SH-IN 4F cells are partially reprogrammed NB cells, and could be a suitable model for investigating the plasticity of aggressive tumors.

Neuroblastoma is a pediatric solid tumor that originates from embryonic neural crest cells. The MYCN gene locus is frequently amplified in unfavorable neuroblastomas, and the gene product promotes the progression of neuroblastomas. However, the molecular mechanisms by which MYCN amplification contributes to stem cell-like states of neuroblastoma remain elusive. In this study, we show that MYCN and its cis-antisense gene, NCYM, form a positive feedback loop with OCT4, a core regulatory gene maintaining a multipotent state of neural stem cells. We previously reported that NCYM is co-amplified with the MYCN gene in primary human neuroblastomas and that the gene product promotes aggressiveness of neuroblastoma by stabilization of MYCN. In 36 MYCN-amplified primary human neuroblastomas, OCT4 mRNA expression was associated with unfavorable prognosis and was correlated with that of NCYM. The OCT4 protein induced both NCYM and MYCN in human neuroblastoma cells, whereas NCYM stabilized MYCN to induce OCT4 and stem cell-related genes, including NANOG, SOX2, and LIN28. In sharp contrast to MYCN, enforced expression of c-MYC did not enhance OCT4 expression in human neuroblastoma cells. All-trans retinoic acid treatment reduced MYCN, NCYM, and OCT4 expression, accompanied by the decreased amount of OCT4 recruited onto the intron 1 region of MYCN. Knockdown of NCYM or OCT4 inhibited formation of spheres of neuroblastoma cells and promoted asymmetric cell division in MYCN-amplified human neuroblastoma cells. These results suggest that the functional interplay between MYCN, NCYM, and OCT4 contributes to aggressiveness of MYCN-amplified human neuroblastomas.

NCYM is a cis-antisense gene of MYCN and is amplified in human neuroblastomas. High NCYM expression is associated with poor prognoses, and the NCYM protein stabilizes MYCN to promote proliferation of neuroblastoma cells. However, the molecular mechanisms of NCYM in the regulation of cell survival have remained poorly characterized. Here we show that NCYM promotes cleavage of MYCN to produce the anti-apoptotic protein, Myc-nick, both in vitro and in vivo. NCYM and Myc-nick were induced at G2/M phase, and NCYM knockdown induced apoptotic cell death accompanied by Myc-nick downregulation. These results reveal a novel function of NCYM as a regulator of Myc-nick production in human neuroblastomas. (C) 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).