Regulation of Tumor Metabolism by Retinoblastoma Protein

描述(申请人提供)：肿瘤发生不仅需要失去增殖控制，而且需要代谢转变为增加葡萄糖和谷氨酰胺消耗，以增加能量产生和推动细胞分裂所必需的核苷酸、氨基酸和脂类的从头合成。这些过程主要是通过癌基因或肿瘤抑制基因的缺失来驱动的，它们绕过了正常的调控途径。视网膜母细胞瘤(Rb)蛋白是最早被发现的肿瘤抑制因子，广泛参与细胞周期调控，在大多数肿瘤类型中都存在Rb通路的干扰。除细胞周期调控外，Rb还参与多种已知参与肿瘤进展的其他生化途径，如转移和血管生成。然而，人们对Rb在调节人类癌症中观察到的独特新陈代谢变化中的作用知之甚少。利用稳定的13C-葡萄糖同位素异构体核磁共振分析发现，与野生型(WT)成纤维细胞相比，小鼠胚胎成纤维细胞(MEF)中RB家族三个成员的三重敲除(TKO)导致葡萄糖摄取和乳酸通量增加，同时减少葡萄糖衍生碳进入TCA循环中间体。这种代谢变化是由多种糖酵解酶的表达变化所介导的，包括GLUT-1、HK2、PK-M2水平的增加和HK1、ALT1和PCb的下降。为了补充TCA循环中葡萄糖碳的损失，我们推测Rb TKO MEF可能会增加生物能量和合成代谢前体对谷氨酰胺的摄取。我们观察到，Rb的缺失通过增加谷氨酰胺转运体ASCT2的表达和谷氨酰胺酶1(GLS1)的活性，增加了13C-谷氨酰胺的摄取和流向谷氨酸和TCA循环中间体。此外，谷氨酰胺碳能够促进氧的消耗，而谷氨酰胺停用导致TKO细胞的ATP水平显著下降。重要的是，这种向谷氨酰胺利用的转变对于RB TKO MEF的生存是必不可少的，而对WT MEF来说并不是，外源性β-酮戊二酸的加入能够挽救RB耗竭细胞中的ATP水平和细胞活力。从机制上讲，E2F-1、-2和-3在功能上改变了葡萄糖和谷氨酰胺的摄取，并且观察到E2F-1和-3与GLUT-1和ASCT2的启动子直接相关。综上所述，这些研究表明，Rb/E2F级联直接调节肿瘤生长所必需的两条关键代谢途径。我们假设，人类癌症中Rb蛋白的失活导致全球代谢转向糖酵解和谷氨酰胺利用，这反过来又是肿瘤永生化和转化所必需的。我们将通过以下具体目标来验证这一假设：1.确定Rb缺失所调控的精确代谢转运体、酶和途径，以及这些代谢靶点对Rb熟练和Rb缺乏的MEF、人类正常上皮细胞和人类癌细胞生存和生长的相对要求。2.探讨Rb1基因缺失对肺腺癌体内葡萄糖/谷氨酰胺代谢及生长的影响。3.探讨人肺癌体内Rb功能丧失与~(13)C-葡萄糖利用变化的关系。

DESCRIPTION (provided by applicant): Tumorigenesis requires not only loss of proliferative control, but also a metabolic shift towards increased glucose and glutamine consumption required for increased energy production and to drive the de novo biosynthesis of nucleotides, amino acids, and lipids essential for cell division. These processes are primarily driven through oncogenes or loss of tumor suppressors, which act to circumvent normal regulatory pathways. The retinoblastoma (Rb) protein, the first described tumor suppressor, is extensively involved in cell cycle regulation, and perturbations within the Rb pathway are found in most tumor types. Beyond cell cycle control, Rb has been implicated in multiple additional biochemical pathways known to be involved in tumor progression, such as metastasis and angiogenesis. However, little is known about the role of Rb in regulating the unique changes in metabolism that have been observed in human cancers. Using stable 13C-glucose isotopomer NMR analyses, we found that triple knock-out (TKO) of all three Rb family members in mouse embryonic fibroblasts (MEFs) resulted in increased glucose uptake and flux to lactate, and simultaneously decreased glucose-derived carbon incorporation into TCA cycle intermediates relative to wild-type (WT) MEFs. This metabolic shift was mediated by changes in the expression of multiple glycolytic enzymes including increased Glut-1, HK2, PK-M2 levels and decreased HK1, ALT1, and PCB. To supplement this loss of glucose carbons for anaplerosis within the TCA cycle, we speculated that the Rb TKO MEFs may increase glutamine uptake for both bioenergetic and anabolic precursors. We observed that loss of Rb caused increased 13C- glutamine uptake and flux into glutamate and TCA cycle intermediates via enhanced expression of the glutamine transporter ASCT2 and the activity of glutaminase 1 (GLS1). Further, glutamine carbon is capable of facilitating oxygen consumption, and glutamine withdrawal resulted in significant decrease in ATP levels in the TKO cells. Importantly, this shift towards glutamine utilization was essential fo the survival of Rb TKO MEFs and not for the WT MEFs, and addition of exogenous ?-ketoglutarate was able to rescue both ATP levels and cell viability in the Rb-depleted cells. Mechanistically, E2F-1, -2, & -3 functionally alter both glucose and glutamine uptake, and E2F-1 & -3 were observed to directly associate with the promoters of Glut-1 and ASCT2. Combined, these studies suggest that the Rb/E2F cascade directly regulates two key metabolic pathways that are necessary for neoplastic growth. We hypothesize that inactivation of the Rb protein in human cancers leads to a global metabolic shift towards enhanced glycolysis and glutamine utilization, which in turn is required for neoplastic immortalization and transformation. We will test this hypothesis by conducting the following Specific Aims: 1. To determine the precise metabolic transporters, enzymes and pathways that are modulated by the loss of Rb and the relative requirements of these metabolic targets for the survival and growth of Rb-proficient and Rb-deficient MEFs, human normal epithelial cells and human cancer cells. 2. To determine the effects of Rb1 deletion on glucose/glutamine metabolism and growth of lung adenocarcinomas in vivo. 3. To correlate the loss of Rb function with changes in the 13C-glucose utilization by human lung tumors in vivo.