PRC2 molecule EED is a target of epigenetic therapy of neuroblastoma

Epigenetic modification by polycomb repressive complex (PRC) molecules appears to have a role in tumorigenesis and aggressiveness of neuroblastoma (NB). Embryonic Ectoderm Development (EED) is a member of PRC2 complex and binds the H3K27me3 mark deposited by EZH2, via propagation on adjacent nucleosomes. Here we studied the molecular roles of EED in MYCN-amplified neuroblastoma cells by using EED-knocked down shRNAs, EED-knocked out sgRNAs, and EED small molecule inhibitor EED226. EED suppression profoundly inhibited the NB cell proliferation and flat-and soft agar colony formation. Transcriptome analysis by microarray of the EED-KD NB cells indicated the de-repression of the cell cycle regulated and differentiation-related genes; GSEA analysis results suggested that cell cycle repressed gene sets were strongly upregulated. Further, epigenetic treatment by the combination of EED inhibitor EED226 and HDAC inhibitor valproic acid effectively suppressed NB cell proliferation and colony formation. The combinatory epigenetic treatment up-regulated the cell cycle regulation- and differentiation-related genes. RNA was extracted at 10 days after shEED-expressing lentiviral infection, and a microarray analysis was performed using the Agilent platform of 8x60 K design ID G4851B (Agilent Single Color. 39494, Agilent Technologies). Two hundred nanograms of total RNA was labeled with Cyanine3 using a Low Input Quick-Amp Labeling Kit (one color, Agilent Technologies) according to the manufacturer’s instructions. Purified labeled total RNA was hybridized to the expression microarray. Hybridization, the scanning of microarrays, and data extraction from scanned images were conducted according to Agilent protocol version 6.9.