Polycomb EZH1 regulates cell cycle/5-fluorouracil sensitivity of neuroblastoma cells in concert with MYCN.

In the present study, we found that EZH1 depletion in MYCN-amplified neuroblastoma cells resulted in significant cell death as well as xenograft inhibition. EZH1 depletion decreased the level of H3K27me1; the interaction and protein stabilization of MYCN and EZH1 appear to play roles in epigenetic transcriptional regulation. Transcriptome analysis of EZH1-depleted cells resulted in downregulation of the cell cycle progression-related pathway. In particular, Gene Set Enrichment Analysis revealed downregulation of reactome E2F-mediated regulation of DNA replication along with key genes of this process, TYMS, POLA2, and CCNA1. TYMS and POLA2 were transcriptionally activated by MYCN and EZH1-related epigenetic modification. Treatment with the EZH1/2 inhibitor UNC1999 also induced cell death, decreased H3K27 methylation, and reduced the levels of TYMS in neuroblastoma cells. Previous reports indicated neuroblastoma cells are resistant to 5-fluorouracil (5-FU) and TYMS (encoding thymidylate synthetase) has been considered the primary site of action for folate analogues. Intriguingly, UNC1999 treatment significantly sensitized MYCN-amplified neuroblastoma cells to 5-FU treatment, suggesting that EZH inhibition could be an effective strategy for development of a new epigenetic treatment for neuroblastoma.

Optimization of surgical timing of congenital diaphragmatic hernia using the quantified flow patterns of patent ductus arteriosus.

PURPOSE: The optimal timing of surgery for congenital diaphragmatic hernia (CDH) is controversial. We aimed to validate our protocol for the timing of CDH repair using the quantified patent ductus arteriosus (PDA) flow pattern. METHODS: This retrospective comparative study analyzed patients with a prenatal diagnosis of isolated CDH between 2007 and 2020. We defined the "LR ratio" as the percentage of velocity-time integral (VTI) of the left-to-right flow of PDA against overall VTI on echocardiography. Since 2010, we followed the decision criterion of performing surgery when LR ratio of > 50% has been achieved in the patients (protocol group). The protocol group (2010-2020) was compared with the historical control group (2007-2009). RESULTS: The average age at surgery was 104.1 ± 175.9 and 37.3 ± 30.6 h in the control and protocol groups, respectively (p = 0.11). Survival rate (88.9% vs. 95.0%, p = 0.53) and the rate of worsening of pulmonary hypertension within 24 h after surgery (22.2% vs. 10.0%, p = 0.57) were not different between the groups. The protocol group had a significantly shorter duration of tracheal intubation (26.9 ± 21.1 vs. 13.3 ± 9.5 days, p = 0.03). CONCLUSION: Our decision criterion might have the advantage of facilitating early and safe surgery for patients with CDH.

EZH2 regulates neuroblastoma cell differentiation via NTRK1 promoter epigenetic modifications.

The polycomb repressor complex 2 molecule EZH2 is now known to play a role in essential cellular processes, namely, cell fate decisions, cell cycle regulation, senescence, cell differentiation, and cancer development/progression. EZH2 inhibitors have recently been developed; however, their effectiveness and underlying molecular mechanisms in many malignancies have not yet been elucidated in detail. Although the functional role of EZH2 in tumorigenesis in neuroblastoma (NB) has been investigated, mutations of EZH2 have not been reported. A Kaplan-Meier analysis on the event free survival and overall survival of NB patients indicated that the high expression of EZH2 correlated with an unfavorable prognosis. In order to elucidate the functional roles of EZH2 in NB tumorigenesis and its aggressiveness, we knocked down EZH2 in NB cell lines using lentivirus systems. The knockdown of EZH2 significantly induced NB cell differentiation, e.g., neurite extension, and the neuronal differentiation markers, NF68 and GAP43. EZH2 inhibitors also induced NB cell differentiation. We performed a comprehensive transcriptome analysis using Human Gene Expression Microarrays and found that NTRK1 (TrkA) is one of the EZH2-related suppression targets. The depletion of NTRK1 canceled EZH2 knockdown-induced NB cell differentiation. Our integrative methylome, transcriptome, and chromatin immunoprecipitation assays using NB cell lines and clinical samples clarified that the NTRK1 P1 and P2 promoter regions were regulated differently by DNA methylation and EZH2-related histone modifications. The NTRK1 transcript variants 1/2, which were regulated by EZH2-related H3K27me3 modifications at the P1 promoter region, were strongly expressed in favorable, but not unfavorable NB. The depletion and inhibition of EZH2 successfully induced NTRK1 transcripts and functional proteins. Collectively, these results indicate that EZH2 plays important roles in preventing the differentiation of NB cells and also that EZH2-related NTRK1 transcriptional regulation may be the key pathway for NB cell differentiation.