Integrating metabolism, apoptosis and the oxidative stress response in cancers

描述（由申请人提供）：癌细胞需要多种适应才能存活。两个关键的适应包括逃避细胞凋亡和代谢重排的能力。尽管这两种途径显然需要相交，但支持这种趋同的证据很少。我们已经观察到小的促凋亡蛋白Noxa能够调节单独的代谢途径，因此可能代表代谢和凋亡之间的交集。我们实验室之前的工作表明，Noxa过表达的细胞增加葡萄糖消耗，减少糖酵解碳通量进入柠檬酸循环（TCA）。表达Noxa的细胞也增加了谷氨酰胺衍生碳进入TCA的通量，独立于上述糖酵解作用。我们之前已经证明，在细胞质中，Noxa在葡萄糖的存在下被磷酸化并招募到大的多蛋白复合物中。在这些复合物中发现Noxa， Mcl-1和糖酵解酶GAPDH的促生存结合伴侣。我们观察到与Mcl-1复合物的GAPDH在响应葡萄糖时以一种可能抑制其糖酵解活性的方式进行翻译后修饰。我们的数据表明Noxa通过Mcl-1和GAPDH间接调节糖酵解。另一方面，线粒体Noxa可能在调节谷氨酰胺和-酮戊二酸如何被利用方面发挥关键作用。我们观察到，在缺乏Noxa的细胞中，谷氨酰胺衍生的TCA中间体¿-酮戊二酸在线粒体中积累，增加线粒体呼吸，导致大量细胞凋亡。重要的是，Noxa的这些线粒体代谢作用独立于其凋亡功能，因为缺乏凋亡的突变体挽救了对¿-酮戊二酸盐的敏感性。本文提出的研究将通过调查Noxa对上述两种途径的调节机制来解决Noxa在代谢中的广泛作用。Specific Aim 1将研究Mcl-1/GAPDH/Noxa轴的代谢后果。蛋白质复合物，特别是Mcl-1和GAPDH之间的相互作用将通过遗传操作被破坏，生化分析和靶向代谢组学将用于询问这种破坏的代谢影响。特异性目标2将研究Noxa在线粒体-酮戊二酸利用中的作用，重点是苹果酸/天冬氨酸穿梭。在缺乏Noxa或存在其非凋亡突变体的情况下，将研究特定代谢物治疗对细胞致敏或修复苹果酸/天冬氨酸穿梭中潜在缺陷的能力，以了解Noxa如何影响这一途径。当我们通过Bcl-2家族蛋白Noxa检查这些途径的趋同时，这些研究结果将为癌症中细胞凋亡逃避和代谢改变提供有价值的见解。更重要的是，了解细胞凋亡和代谢重排之间的串扰可能会导致发现能够同时破坏癌症的两个关键支柱的治疗靶点。

DESCRIPTION (provided by applicant): Cancer cells require several adaptations to survive. Two key adaptations include the ability to evade apoptosis and metabolic rearrangement. Despite an obvious requirement for these two pathways to intersect, very little evidence exists supporting this convergence. We have observed that the small pro-apoptotic protein, Noxa, is capable of regulating separate metabolic pathways and therefore may represent an intersection between metabolism and apoptosis. Previous work in our lab showed that Noxa overexpressing cells increase glucose consumption and decrease glycolytic carbon flux into the citric acid cycle (TCA). Noxa expressing cells also increase the flux of glutamine derived carbon into the TCA, independent of the glycolytic role described above. We had shown previously that in the cytoplasm Noxa is phosphorylated and recruited into large multi- protein complexes in the presence of glucose. Found within these complexes is the pro-survival binding partner of Noxa, Mcl-1, and the glycolytic enzyme GAPDH. We observed that GAPDH in complex with Mcl-1 is post-translationally modified in response to glucose in a manner that is likely to inhibit its glycolytic activity. Our data suggest Noxa regulates glycolysis indirectly through Mcl-1 and GAPDH. Mitochondrial Noxa, on the other hand, may play a critical role in regulating how glutamine and ¿-ketoglutarate are utilized. We observed that in cells lacking Noxa, the glutamine derived TCA intermediate ¿-ketoglutarate accumulated in the mitochondria and increased mitochondrial respiration prior to causing massive apoptosis. Importantly, these mitochondrial metabolic effects of Noxa were independent of its apoptotic function, as a mutant deficient for apoptosis rescued sensitivity to ¿-ketoglutarate. The studies proposed here will address Noxa's broad role in metabolism by investigating the mechanism underlying its regulation of the two pathways described above. Specific Aim 1 will investigate the metabolic consequences of the Mcl-1/GAPDH/Noxa axis. Protein complexes and, specifically, interactions between Mcl-1 and GAPDH will be disrupted by genetic manipulation, and biochemical assays and targeted metabolomics will be used to interrogate the metabolic effects of the disruption. Specific Aim 2 will examine Noxa's role in mitochondrial ¿-ketoglutarate utilization, focusing on the malate/aspartate shuttle. The ability of specific metabolite treatments to sensitize cells or t rescue potential deficiencies in the malate/aspartate shuttle, either in the absence of Noxa or in the presence of its non-apoptotic mutant, will be examined to understand how Noxa affects this pathway. The results of these studies will offer valuable insight into both apoptosis evasion and metabolic alterations in cancer as we examine the convergence of these pathways through the Bcl-2 family protein, Noxa. More significantly, understanding the crosstalk between apoptosis and metabolic rearrangement could lead to the discovery of therapeutic targets capable of simultaneously disrupting two critical pillars of cancer.