Regulation of Tumor Metabolism by Retinoblastoma Protein

视网膜母细胞瘤蛋白对肿瘤代谢的调节

• 批准号: 9065696
• 负责人: Brian F Clem
• 金额: $ 19.58万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9065696
• 关键词: ATP Synthesis Pathway; Affect; Alanine Transaminase; Amino Acids; Antineoplastic Agents; Biochemical Pathway; Bioenergetics; Biological Markers; Cancer Etiology; Carbon; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Survival; Cell division; Cells; Characteristics; Citric Acid Cycle; Complex; Consumption; Data; Development; E2F transcription factors; E2F1 gene; Embryo; Enzymes; Epithelial Cells; Family member; Fibroblasts; Functional disorder; Gene Expression; Genes; Genomics; Glucose; Glutamates; Glutaminase; Glutamine; Glycolysis; Growth; HK2 gene; Health; Histones; Human; Knock-out; Knockout Mice; Lipids; Lung Adenocarcinoma; Lung Neoplasms; Malignant Neoplasms; Mediating; Metabolic; Metabolic Pathway; Metabolism; Molecular Target; Mus; Neoplasm Metastasis; Neoplasms; Neoplastic Cell Transformation; Normal Cell; Normal tissue morphology; Nucleotide Biosynthesis; Oncogenes; Oxygen Consumption; Pathway interactions; Polychlorinated Biphenyls; Positron-Emission Tomography; Process; Production; Promoter Regions; Proteins; Recruitment Activity; Regulation; Regulatory Pathway; Retinoblastoma; Retinoblastoma Protein; Role; TOCSY; Testing; Transcription Repressor/Corepressor; Tumor Suppressor Proteins; Wild Type Mouse; Withdrawal; alpha ketoglutarate; angiogenesis; base; bcr-abl Fusion Proteins; c-myc Genes; cancer cell; cell transformation; chromatin remodeling; enzyme pathway; glucose uptake; in vivo; macromolecule; metabolic phenotype; mitochondrial metabolism; neoplastic; neoplastic cell; potential biomarker; promoter; small molecule; tumor; tumor initiation; tumor metabolism; tumor progression; tumorigenesis; uptake

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 α-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.

描述（由适用提供）：肿瘤发生不仅需要扩散控制的丧失，而且还需要向增加能量产生所需的葡萄糖和谷氨酰胺消耗的代谢转变，并驱动核动肽，氨基酸，氨基酸和细胞分裂必不可少的脂质的新生生物合成。这些过程主要是通过肿瘤基因或肿瘤补充剂的损失来驱动的，这些肿瘤补充剂起作用以规避正常的调节途径。视网膜母细胞瘤（RB）蛋白是第一个描述的肿瘤抑制剂，广泛参与细胞周期调节，并且在大多数肿瘤类型中发现RB途径内的扰动。除了细胞周期控制之外，RB在已知参与肿瘤进展的多种其他生化途径中也暗示了RB，例如转移和血管生成。但是，对于使用稳定的13C-葡萄糖同位素NMR分析的RB在调节新陈代谢中的独特变化中的作用知之甚少，我们发现所有三个RB家族成员在小鼠胚胎成纤维细胞（MEF）中的三个RB家族成员的三重敲除（TKO）导致了增加的糖含量和葡萄糖量，并逐渐掺入了糖尿酸盐，并将其掺入了糖尿酸盐量，并逐渐掺入了糖尿酸盐，并逐渐掺入了糖尿含量的胶状效果。中间相对于野生型（WT）MEF。这种代谢转移是由多种糖酵解酶表达的变化介导的，包括增加GLUT-1，HK2，PK-M2水平以及HK1，ALT1和PCB的增加。为了补充TCA周期内葡萄糖碳的损失，我们推测RB TKO MEF可能会增加生物能和合成代谢前体的谷氨酰胺摄取。我们观察到，RB的损失通过增强的谷氨酰胺转运蛋白ASCT2的表达以及谷氨酰胺1的活性（GLS1）引起了13c-谷氨酰胺吸收和进入谷氨酸和TCA循环中间体的增加。此外，谷氨酰胺碳能够支持消耗氧气，而谷氨酰胺的戒断导致TKO细胞中ATP水平的显着降低。重要的是，朝谷氨酰胺利用率的转变是RB TKO MEF的生存至关重要的，而不是WT MEF的生存，而添加外源α-酮戊二酸的添加能够挽救RB缺失细胞中ATP水平和细胞生存能力。从机械上讲，E2F -1，-2和-3在功能上会改变葡萄糖和谷氨酰胺的摄取，并且观察到E2F -1和-3直接与Glut -1和ASCT2的启动子直接相关。这些研究结合在一起，表明RB/E2F级联直接调节了两种对于肿瘤生长所必需的关键代谢途径。我们假设人类癌症中RB蛋白的灭活导致全球代谢转向增强的糖酵解和谷氨酰胺的利用，这又是肿瘤的永生化和转化所必需的。我们将通过执行以下特定目的来检验这一假设：1。确定由RB丢失以及这些代谢靶标对RB功能和RB缺乏型MEF的生存和生长的相对要求所调节的精确代谢转运蛋白，酶和途径，这些靶标的和RB缺乏的MEF，人类正常的上皮细胞和人类上皮细胞和人类癌细胞。 2。确定RB1缺失对体内葡萄糖/谷氨酰胺代谢的影响和肺腺癌的生长。 3。将RB功能的损失与人体肺肿瘤在体内的13C-葡萄糖利用率的变化相关联。