Targeting Glucose Metabolism in Cancer

靶向癌症中的葡萄糖代谢

• 批准号: 7699623
• 负责人: Sucheta Telang
• 金额: $ 24.57万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7699623
• 关键词: 6-Phosphofructokinase; A549; Actins; Adenocarcinoma Cell; Age-Months; Anchorage-Independent Growth; Apoptosis; Attenuated; Basement membrane; Binding; Brain; Breast; Carbon; Cell Fractionation; Cells; Chickens; Citric Acid Cycle; Colon; Cytoplasm; Data; Development; Enzymes; Epithelial Cells; Exons; Family; Freezing; Fructose; Gel; Genes; Genomics; Genus Cola; Glucose; Glycolysis; Glycolysis Inhibition; Growth; Heart; Human; Hypoxia; Immunohistochemistry; In Situ; In Situ Nick-End Labeling; In Vitro; Investigation; Isoenzymes; Knockout Mice; Label; Lactic acid; Lamins; Large T Antigen; Liver; Lung; Lung Adenocarcinoma; Lung Neoplasms; Malignant Neoplasms; Mammary Neoplasms; Mammary gland; Measures; Messenger RNA; Metabolic; Metabolism; Mitochondria; Modeling; Mus; Mutation; NADH; Normal tissue morphology; Nude Mice; Oncogenic; Open Reading Frames; Organ; Ovary; Oxygen; Oxygen Consumption; Pathway interactions; Phenotype; Phosphoric Monoester Hydrolases; Phosphotransferases; Placenta; Positron-Emission Tomography; Production; Protein p53; Proteins; Regulation; Relative (related person); Reporting; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Role; Screening procedure; Shunt Device; Site; Skeletal Muscle; Small Interfering RNA; Source; Specimen; Subfamily lentivirinae; TP53 gene; Tamoxifen; Telomerase; Testing; Testis; Tissues; Transgenes; Tumor Tissue; Vascularization; Western Blotting; base; cancer cell; cancer type; cell transformation; cellular transduction; design; glucose metabolism; glucose uptake; in vivo; inhibitor/antagonist; mRNA Expression; mortality; neoplastic; neoplastic cell; neurotensin mimic 1; novel; overexpression; promoter; protein expression; public health relevance; recombinase; small hairpin RNA; small molecule; tumor; tumor growth; vector

DESCRIPTION (provided by applicant): A high rate of glycolytic flux, even in the presence of oxygen, is a central metabolic hallmark of neoplastic tumors. Cancer cells preferentially utilize glycolysis in order to satisfy their increased energetic and biosynthetic requirements. This metabolic phenotype is confirmed by positron emission tomography (PET) with 2-[18F]-fluoro-2-deoxy-glucose which demonstrates that tumors take up 10-fold more glucose than adjacent normal tissues in vivo. Over-expression of HIF-1a and myc, ras activation and loss of p53 function stimulate glycolysis by activating a family of four bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB) which synthesize fructose-2,6-bisphosphate (F2,6BP), an allosteric activator of 6-phosphofructo-1- kinase (PFK-1) which is an essential control point in the glycolytic pathway. The PFKFB enzymes are encoded by four genes (PFKFB1-4) reportedly expressed in distinct tissues: liver/skeletal muscle (PFKFB1), heart (PFKFB2), placenta (PFKFB3) and testis (PFKFB4). Previous studies have focused on the PFKFB3 isozyme as the dominant source of F2,6BP in neoplastic cells due to its high kinase:phosphatase ratio (~740:1) and overexpression in multiple tumors. The identification of the specific PFKFB isozyme responsible for the high rate of glycolysis in cancer cells may allow for the development of novel agents that suppress tumor growth through inhibition of glycolysis. In preliminary studies, we measured mRNA expression of the four PFKFB isozymes in 20 tumor tissues by multiplex RT-PCR and found that PFKFB4 mRNA (and not PFKFB3) was markedly increased in 17/20 tumors relative to adjacent, normal tissues. We speculated that the high PFKFB4 expression may be caused by disparate oncogenic pathways converging to enhance glycolytic flux. We found that hypoxic exposure of K-rasG12S+ A549 lung adenocarcinoma cells and introduction of H-rasG12V into immortalized human bronchial epithelial cells increased PFKFB4 mRNA and protein expression. Further, cell fractionation revealed that the PFKFB4 protein, and not PFKFB3, localized to the cytoplasm, the cellular compartment of both PFK-1 and glycolysis. Last, transient siRNA silencing of PFKFB4 mRNA expression in K- rasG12S+ A549 cells decreased steady-state concentration of F2,6BP, glycolytic flux and anchorage- independent tumor growth in mice. Taken together, these preliminary studies provide rationale for further investigation of the PFKFB4 isozyme as a possible novel target for the development of anti-neoplastic agents. We hypothesize that the PFKFB4 isozyme is required for the high glycolytic flux, survival, growth and spread of transformed cells. We plan to test this hypothesis by pursuing the following specific aims: 1. To examine the requirement for F2,6BP synthesized by PFKFB4 for glycolytic and mitochondrial metabolism. 2. To determine the requirement for F2,6BP synthesized by PFKFB4 for growth, invasiveness and survival of normal, immortalized and ras-transformed epithelial cells. 3. To examine the effect of PFKFB4 genomic deletion on growth and metabolism of ras-dependent tumors in vivo using a Cre-lox inducible mouse knockout of PFKFB4. PUBLIC HEALTH RELEVANCE: We anticipate that shRNA silencing or genomic deletion of the PFKFB4 isozyme will attenuate the neoplastic potential of transformed epithelial cells and conversely, that increased F2,6BP (from overexpression of PFKFB4) will enhance glycolytic flux and augment tumor growth. These findings may validate PFKFB4 as a new target for the inhibition of glycolysis. Accordingly, suppression of glycolytic flux in cancer via the development of small molecule inhibitors of PFKFB4 through computational screening for competitive inhibitors of the PFKFB4 substrate-binding domain may reduce the high mortality that is associated with cancer.

描述（由申请人提供）：即使存在氧气，高糖酵解通量也是肿瘤肿瘤的中心代谢标志。癌细胞优先利用糖酵解，以满足其能量和生物合成需求的增加。这种代谢表型通过具有2- [18F] -Fluoro-2-脱氧 - 葡萄糖的正电子发射断层扫描（PET）证实，这表明肿瘤比体内相邻正常组织的葡萄糖占10倍。 HIF-1A和MYC的过表达，RAS激活和p53功能的丧失通过激活四个双功能的6-磷酸2-激酶/果糖-2,6-磷酸酶（PFKFB）的家族，从而刺激糖酵解激酶（PFK-1）是糖酵解途径中的重要控制点。据报道，PFKFB酶由四个基因（PFKFB1-4）编码，据报道在不同的组织中表达：肝脏/骨骼肌（PFKFB1），心脏（PFKFB2），胎盘（PFKFB3）（PFKFB3）和睾丸（PFKFB4）（PFKFB4）。先前的研究集中在PFKFB3同工酶作为肿瘤细胞中F2,6bp的主要来源，这是由于其高激酶：磷酸酶比（〜740：1）和多种肿瘤中的过表达。鉴定癌细胞中高糖酵解速率的特异性PFKFB同工酶可能会允许通过抑制糖酵解来抑制肿瘤生长的新型药物的发展。在初步研究中，我们通过多重RT-PCR测量了20个肿瘤组织中四个PFKFB同工酶的mRNA表达，发现相对于相对于邻近正常组织的17/20个肿瘤中的PfKFB4 mRNA（而非PFKFB3）显着增加。我们推测，高PFKFB4表达可能是由不同的致癌途径融合以增强糖酵解通量引起的。我们发现，K-RASG12S+ A549肺腺癌细胞的低氧暴露以及将H-Rasg12V引入永生的人支气管上皮细胞中增加了PFKFB4 mRNA和蛋白质的表达。此外，细胞分馏表明，PFKFB4蛋白，而不是PFKFB3，该蛋白位于细胞质，即PFK-1和糖酵解的细胞区室。最后，在K-RASG12S+ A549细胞中PFKFB4 mRNA表达的瞬时siRNA沉默降低了小鼠F2,6bp的稳态浓度，糖酵解通量和锚固独立的肿瘤生长。综上所述，这些初步研究为进一步研究PFKFB4同工酶提供了基本原理，这是抗塑性剂的发展的可能的新靶标。我们假设PFKFB4同工酶是高糖酵解通量，转化细胞的生存，生长和扩散所必需的。我们计划通过追求以下特定目的来检验这一假设：1。检查PFKFB4对糖酵解和线粒体代谢合成的F2,6bp的要求。 2。确定PFKFB4合成的F2,6bp对正常，永生和RAS转换上皮细胞的生长，侵入性和存活的要求。 3。使用PFKFB4的Cre-Lox诱导小鼠敲除体内RAS依赖性肿瘤的生长和代谢的影响。公共卫生相关性：我们预计PFKFB4同工酶的shRNA沉默或基因组缺失将减弱转化的上皮细胞的肿瘤潜力，相反，增加了F2,6BP（来自PFKFB4的过表达）将增强糖脂溶解和增强的肿瘤肿瘤的增长。这些发现可能会验证PFKFB4作为抑制糖酵解的新目标。因此，通过计算筛查PFKFB4底物结合结构域的竞争性抑制剂来抑制癌症中的糖酵解通量，可以降低与癌症相关的高死亡率。