Integrating metabolism, apoptosis and the oxidative stress response in cancers

描述（由申请人提供）：癌细胞需要几种适应才能生存。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交叉，但很少有证据支持这种趋同。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叉。我们实验室以前的工作表明，Noxa过表达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的细胞还增加谷氨酰胺衍生的碳进入TCA的通量，与上述糖酵解作用无关。我们先前已经表明，在细胞质中，Noxa在葡萄糖存在下被磷酸化并被募集到大的多蛋白复合物中。在这些复合物中发现的是Noxa、Mcl-1和糖酵解酶GAPDH的促存活结合伴侣。我们观察到GAPDH与Mcl-1复合后，以可能抑制其糖酵解活性的方式响应于葡萄糖而被后修饰。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<$-酮戊二酸的利用方面发挥关键作用。我们观察到，在缺乏Noxa的细胞中，谷氨酰胺衍生的TCA中间体<$-酮戊二酸在线粒体中积累，并在引起大量细胞凋亡之前增加线粒体呼吸。重要的是，Noxa的这些线粒体代谢作用不依赖于其凋亡功能，因为凋亡缺陷的突变体挽救了对<$-酮戊二酸的敏感性。本文提出的研究将通过研究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。具体目标1将研究Mcl-1/GAPDH/Noxa轴的代谢结果。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，生物化学测定和靶向代谢组学将用于询问破坏的代谢效应。具体目标2将研究Noxa在线粒体戊酮利用中的作用，重点是苹果酸/天冬氨酸穿梭。在不存在Noxa或存在其非凋亡突变体的情况下，将检查特定代谢物处理使细胞致敏或拯救苹果酸/天冬氨酸穿梭中的潜在缺陷的能力，以了解Noxa如何影响该途径。这些研究的结果将提供有价值的洞察到细胞凋亡逃避和癌症的代谢改变，因为我们检查这些途径的收敛通过Bcl-2家族蛋白，Noxa。更重要的是，了解细胞凋亡和代谢重排之间的相互作用可能会导致发现能够同时破坏癌症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.