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: 8438598
• 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/8438598
• 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; Histones; Human; Knock-out; Knockout Mice; Lipids; Lung Adenocarcinoma; Lung Neoplasms; Malignant Neoplasms; Mediating; Metabolic; Metabolic Pathway; Metabolism; Mitochondria; Molecular Target; Mus; Neoplasm Metastasis; Neoplasms; Normal Cell; Normal tissue morphology; Nucleotide Biosynthesis; Oncogenes; Oxygen Consumption; Pathway interactions; Phenotype; Polychlorinated Biphenyls; Positron-Emission Tomography; Process; Production; Proteins; Recruitment Activity; Regulation; Regulatory Pathway; Relative (related person); Retinoblastoma; Retinoblastoma Protein; Role; TOCSY; Testing; Transcription Repressor/Corepressor; Tumor Suppressor Proteins; Wild Type Mouse; Withdrawal; angiogenesis; base; bcr-abl Fusion Proteins; c-myc Genes; cancer cell; cell transformation; chromatin remodeling; enzyme pathway; glucose uptake; in vivo; macromolecule; neoplastic; neoplastic cell; promoter; public health relevance; 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 ?-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（由申请人提供）：肿瘤发生不仅需要增殖控制的丧失，而且需要代谢向增加的葡萄糖和谷氨酰胺消耗转变，这是增加能量产生和驱动细胞分裂所必需的核苷酸、氨基酸和脂质的从头生物合成所必需的。这些过程主要是通过癌基因或肿瘤抑制因子的缺失来驱动的，其作用是绕过正常的调节途径。视网膜母细胞瘤（Rb）蛋白是第一个被描述的肿瘤抑制因子，广泛参与细胞周期调控，并且在大多数肿瘤类型中发现Rb通路内的扰动。除了细胞周期控制之外，Rb还参与了已知参与肿瘤进展的多种其他生化途径，如转移和血管生成。然而，关于Rb在调节人类癌症中观察到的代谢独特变化中的作用知之甚少。使用稳定的13 C-葡萄糖同位素NMR分析，我们发现，三重敲除（TKO）的所有三个Rb家族成员在小鼠胚胎成纤维细胞（MEFs）导致增加的葡萄糖摄取和乳酸通量，同时减少葡萄糖衍生的碳掺入TCA循环中间体相对于野生型（WT）MEFs。这种代谢转变是由多种糖酵解酶表达的变化介导的，包括Glut-1、HK 2、PK-M2水平升高和HK 1、ALT 1和PCB水平降低。为了补充TCA循环中回补的葡萄糖碳损失，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入。我们观察到Rb的缺失通过增强谷氨酰胺转运体ASCT 2的表达和谷氨酰胺酶1（GLS 1）的活性引起13 C-谷氨酰胺摄取和流入谷氨酸和TCA循环中间体的增加。此外，谷氨酰胺碳能够促进氧消耗，并且谷氨酰胺戒断导致TKO细胞中ATP水平的显著降低。重要的是，这种向谷氨酰胺利用的转变对于Rb TKO MEFs的存活是必不可少的，而不是对于WT MEFs，并且添加外源性？酮戊二酸能够拯救Rb耗尽细胞中的ATP水平和细胞活力。在机制上，E2 F-1、E2 F-2和E2 F-3在功能上改变葡萄糖和谷氨酰胺摄取，并且观察到E2 F-1和E2 F-3与Glut-1和ASCT 2的启动子直接相关。结合，这些研究表明，Rb/E2 F级联直接调节肿瘤生长所必需的两个关键代谢途径。我们假设，在人类癌症中的Rb蛋白的失活导致全球代谢转向增强糖酵解和谷氨酰胺的利用，这反过来又是肿瘤永生化和转化所需的。我们将通过以下具体目标来检验这一假设：1.确定Rb缺失所调控的精确代谢转运蛋白、酶和途径，以及这些代谢靶点对Rb-活性和Rb-缺陷MEFs、人正常上皮细胞和人癌细胞存活和生长的相对需求。2.探讨Rb 1基因缺失对肺腺癌细胞葡萄糖/谷氨酰胺代谢及生长的影响。3. Rb功能的丧失与体内人肺肿瘤对13 C-葡萄糖利用的变化相关。