The physiological functions of butyrylcholinesterase (BChE) and its role in malignancy remain unexplained. Our studies in children newly diagnosed with neuroblastoma indicated that BChE expressions is proportional to MYCN amplification suggesting that pathogenesis of high-risk disease may be related to the persistent expression of abnormally high levels of tumor-associated BChE. BChE-deficient neuroblastoma cells (KO [knockout]) were produced from MYCN-amplified BE(2)-C cells (WT [wild-type]) by the CRISPR-Cas9 targeted disruption of the BCHE locus. KO cells have no detectable BChE activity. The compensatory acetylcholinesterase activity was not detected. The average population doubling time of KO cells is 47.0±2.4 hours, >2× longer than WT cells. Reduced proliferation rates of KO cells were accompanied by the loss of N-Myc protein and a significant deactivation of tyrosine kinase receptors associated with the aggressive neuroblastoma phenotype including Ros1, TrkB, and Ltk. Tumorigenicity of WT and KO cells in male mice was essentially identical. In contrast, KO xenografts in female mice were very small (0.37±0.10 g), ~3× smaller compared with WT xenografts (1.11±0.30 g). Unexpectedly, KO xenografts produced changes in plasma BChE similarly to WT tumors but lesser in magnitude. The disruption of BCHE locus in MYCN-amplified neuroblastoma cells decelerates proliferation and produces neuroblastoma cells that are less aggressive in female mice.

丁酰胆碱酯酶 (BChE) 的生理功能及其在恶性肿瘤中的作用仍不清楚。我们对新诊断为神经母细胞瘤的儿童进行的研究表明，BChE 表达与MYCN扩增成正比，这表明高危疾病的发病机制可能与肿瘤相关 BChE 水平异常高的持续表达有关。通过 CRISPR-Cas9 靶向破坏 BCHE 基因座，从MYCN扩增的 BE(2)-C 细胞（WT [野生型] ）产生BChE缺陷型神经母细胞瘤细胞（KO [敲除]）。KO 细胞没有可检测到的 BChE 活性。未检测到代偿性乙酰胆碱酯酶活性。KO细胞的平均群体倍增时间为47.0±2.4小时，比WT细胞长>2倍。KO 细胞增殖率降低伴随着 N-Myc 蛋白的丢失以及与侵袭性神经母细胞瘤表型相关的酪氨酸激酶受体（包括 Ros1、TrkB 和 Ltk）的显着失活。雄性小鼠中WT和KO细胞的致瘤性基本相同。相比之下，雌性小鼠中的KO异种移植物非常小(0.37±0.10g)，比WT异种移植物(1.11±0.30g)小约3倍。出乎意料的是，KO 异种移植物产生的血浆 BChE 变化与 WT 肿瘤类似，但幅度较小。MYCN扩增的神经母细胞瘤细胞中BCHE位点的破坏会减慢增殖速度，并产生在雌性小鼠中攻击性较低的神经母细胞瘤细胞。