Linking nucleotide and amino acid metabolism to cholesterol synthesis by MYCN

癌基因如何整合代谢途径以满足癌细胞生长和生物合成的需求 增殖是癌症研究的核心问题，具有广泛的临床意义。处于高风险状态 神经母细胞瘤是最致命的儿童癌症之一，甲羟戊酸途径被转录激活。 该代谢途径使用乙酰辅酶A碳来产生胆固醇和其他代谢物，这些代谢物是维持生命所必需的。 维持培养物中神经母细胞瘤细胞系的增殖和神经母细胞瘤肿瘤的生长 鼠标模型。我们最近获得的证据表明，致癌转录因子 MYCN 是 甲羟戊酸途径酶的转录激活和细胞中胆固醇产生的增加 源自具有 MYCN 基因组扩增的高风险神经母细胞瘤的细胞系。拟议的 研究将解决有关 MYCN 重编程作用的分子基础的两个主要问题 甲羟戊酸代谢和胆固醇合成的影响：1) MYCN 如何激活甲羟戊酸途径 增加产量； 2) MYCN 如何协调其他代谢途径以增加乙酰基的供应 辅酶A。在目标 1 中，我们将结合使用细胞和分子方法来检验以下假设： MYCN 通过转录激活破坏甲羟戊酸途径的终产物反馈抑制 SCAP（甲羟戊酸代谢的正调节因子）和 INSIG2（负调节因子）的抑制，导致 甲羟戊酸途径的组成型激活。我们将进一步研究MYCN的分子基础 INSIG2 表达的抑制。在目标 2 中，我们将使用稳定同位素示踪剂与 shRNA 结合使用 沉默和酶抑制剂来描绘供应底物乙酰辅酶A的代谢途径。我们 将检验 MYCN 增加谷氨酰胺碳进入甲羟戊酸途径的通量的假设 通过核苷酸和丝氨酸-甘氨酸合成途径的转录激活来合成胆固醇。瞄准 3、我们将使用稳定同位素示踪剂与酶抑制剂相结合，为 谷氨酰胺作为通过核苷酸和丝氨酸-甘氨酸合成胆固醇合成的主要碳源 患者来源的异种移植物和高危神经母细胞瘤 TH-MYCN 小鼠模型中的通路。 该项目的成功完成将为 MYCN 整合核苷酸、 氨基酸和胆固醇代谢在驱动和维持高风险神经母细胞瘤中的作用，这可能表明 治疗干预的新途径。

How oncogenes integrate metabolic pathways to meet the biosynthetic demands of cancer cell growth and proliferation is a central question of cancer research with broad clinical implications. In high-risk neuroblastoma, one of the deadliest childhood cancers, the mevalonate pathway is transcriptionally activated. This metabolic pathway uses acetyl-CoA carbon to produce cholesterol and other metabolites essential to sustain the proliferation of neuroblastoma cell lines in culture and the growth of neuroblastoma tumors in mouse models. We recently obtained evidence that the oncogenic transcription factor MYCN is required for the transcriptional activation of mevalonate pathway enzymes and the increased production of cholesterol in cell lines derived from high-risk neuroblastoma tumors with genomic amplification of MYCN. The proposed research will address two major questions concerning the molecular basis of MYCN action in reprogramming of mevalonate metabolism and cholesterol synthesis: 1) how MYCN activates the mevalonate pathway to increase its output; and 2) how MYCN coordinates other metabolic pathways to increase the supply of acetyl- CoA. In Aim 1, we will use a combination of cellular and molecular approaches to test the hypothesis that MYCN disrupts end-product feedback inhibition of the mevalonate pathway by transcriptional activation of SCAP, a positive regulator of mevalonate metabolism and repression of INSIG2, a negative regulator, leading to constitutive activation of the mevalonate pathway. We will further investigate the molecular basis for MYCN repression of INSIG2 expression. In Aim 2, we will use stable isotope tracers in combination with shRNA silencing and enzyme inhibitors to delineate the metabolic pathways that supply the substrate acetyl-CoA. We will test the hypothesis that MYCN increases the flux of glutamine carbon into the mevalonate pathway for cholesterol synthesis via transcriptional activation of nucleotide and serine-glycine synthesis pathways. In Aim 3, we will use stable isotope tracers in combination with enzyme inhibitors to provide in vivo evidence for glutamine as a major source of carbon for cholesterol synthesis via nucleotide and serine-glycine synthesis pathways in patient-derived xenografts and the TH-MYCN mouse model of high-risk neuroblastoma. Successful completion of this project will define a molecular mechanism for MYCN to integrate nucleotide, amino acid, and cholesterol metabolism in driving and sustaining high-risk neuroblastoma, which may suggest new avenues of therapeutic intervention.