Supplementary Materials and Methods from PRC2-Mediated Transcriptomic Alterations at the Embryonic Stage Govern Tumorigenesis and Clinical Outcome in MYCN-Driven Neuroblastoma

Tsubota, Shoma; Kishida, Satoshi; Shimamura, Teppei; Ohira, Miki; Yamashita, Satoshi; Cao, Dongliang; Kiyonari, Shinichi; Ushijima, Toshikazu; Kadomatsu, Kenji

doi:10.1158/0008-5472.22414214.v1

00085472can163144-sup-174843_3_supp_4166644_3t310g.pdf (103.6 kB)

Supplementary Materials and Methods from PRC2-Mediated Transcriptomic Alterations at the Embryonic Stage Govern Tumorigenesis and Clinical Outcome in MYCN-Driven Neuroblastoma

journal contribution

posted on 2023-03-31, 00:41 authored by Shoma Tsubota, Satoshi Kishida, Teppei Shimamura, Miki Ohira, Satoshi Yamashita, Dongliang Cao, Shinichi Kiyonari, Toshikazu Ushijima, Kenji Kadomatsu

This file contains additional details of materials and methods used in this study including medium preparation, H&E staining, immunofluorescence analysis of frozen sections, immunohistochemistry on paraffin sections, in situ hybridization, western blotting, qPCR, microarray analysis, array CGH, MBD-seq, ChIP-seq, immunoprecipitation, overexpression of MYCN, knockdown of Ezh2, Ezh2 inhibitor treatment, and statistical analysis.

Funding

JSPS

Japan Agency for Medical Research and Development

CREST

History

ARTICLE ABSTRACT

Pediatric cancers such as neuroblastoma are thought to involve a dysregulation of embryonic development. However, it has been difficult to identify the critical events that trigger tumorigenesis and differentiate them from normal development. In this study, we report the establishment of a spheroid culture method that enriches early-stage tumor cells from TH-MYCN mice, a preclinical model of neuroblastoma. Using this method, we found that tumorigenic cells were evident as early as day E13.5 during embryo development, when the MYC and PRC2 transcriptomes were significantly altered. Ezh2, an essential component of PRC2, was expressed in embryonic and postnatal tumor lesions and physically associated with N-MYC and we observed that H3K27me3 was increased at PRC2 target genes. PRC2 inhibition suppressed in vitro sphere formation, derepressed its target genes, and suppressed in situ tumor growth. In clinical specimens, expression of MYC and PRC2 target genes correlated strongly and predicted survival outcomes. Together, our findings highlighted PRC2-mediated transcriptional control during embryogenesis as a critical step in the development and clinical outcome of neuroblastoma. Cancer Res; 77(19); 5259–71. ©2017 AACR.