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长的生物合成需求 增殖是癌症研究的中心问题，具有广泛的临床意义。处于高风险中 神经母细胞瘤是儿童最致命的癌症之一，甲氧戊酸途径在转录上被激活。 这一代谢途径使用乙酰辅酶A碳来产生胆固醇和其他必需的代谢物 维持神经母细胞瘤细胞系在培养中的增殖和神经母细胞瘤肿瘤的生长 老鼠模型。我们最近获得的证据表明，致癌转录因子MYCN是肿瘤细胞所必需的 甲氧戊酸途径酶的转录激活与细胞内胆固醇生成的增加 来自具有MYCN基因组扩增的高危神经母细胞瘤的株系。建议数 研究将解决关于MYCN在重编程中作用的分子基础的两个主要问题 甲氧丙戊酸代谢和胆固醇合成：1)MYCN如何激活甲氧戊酸途径 增加其产量；以及2)MYCN如何协调其他代谢途径来增加乙酰基的供应- 可口可乐。在目标1中，我们将使用细胞和分子方法的组合来检验假设 MYCN通过转录激活MYCN干扰甲氧丙戊酸途径的最终产物反馈抑制 甲氧丙戊酸代谢的正调控因子SCAP和负性调节因子INSIG2的抑制 甲氧戊酸途径的结构性激活。我们将进一步研究MYCN的分子基础 抑制INSIG2的表达。在目标2中，我们将结合shrna使用稳定的同位素示踪剂。 沉默和酶抑制剂来描绘提供底物乙酰-辅酶A的代谢途径。我们 将检验以下假设：MYCN增加谷氨酰胺碳进入甲羟戊酸途径的流量 通过转录激活核苷酸和丝氨酸-甘氨酸合成途径合成胆固醇。在AIM 3，我们将使用稳定的同位素示踪剂与酶抑制剂相结合，为 谷氨酰胺是通过核苷酸和丝氨酸-甘氨酸合成胆固醇的主要碳源 患者来源的异种移植的途径和高危神经母细胞瘤的TH-MYCN小鼠模型。 该项目的成功完成将确定MYCN整合核苷酸的分子机制， 氨基酸和胆固醇代谢在驱动和维持高危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.