Targeting Glucose Metabolism in Cancer

靶向癌症中的葡萄糖代谢

• 批准号: 8063108
• 负责人: Sucheta Telang
• 金额: $ 17.87万
• 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/8063108
• 关键词: 6-Phosphofructo-2-kinase; 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; 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; Mouse Mammary Tumor Virus; 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; Screening procedure; Site; Skeletal Muscle; Small Interfering RNA; Source; Specimen; Subfamily lentivirinae; TOCSY; 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; shunt pathway; 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)：即使在氧气存在的情况下，糖酵解通量也很高，这是肿瘤的中心代谢标志。癌细胞优先利用糖酵解来满足它们增加的能量和生物合成需求。2-[18F]-氟-2-脱氧葡萄糖的正电子发射断层扫描(PET)证实了这种代谢表型，这表明肿瘤体内摄取的葡萄糖是邻近正常组织的10倍。HIF-1a和myc的过度表达、ras的激活和p53功能的丧失通过激活四个双功能6-phosphofructo-2-kinase/fructose-2，6-bisphosphatases家族来刺激糖酵解，该家族合成果糖-2，6-二磷酸(F2，6BP)，是6-磷酸果糖-1-激酶(Pfk-1)的变构激活剂，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，它们分别在肝脏/骨骼肌(PFKFB1)、心脏(PFKFB2)、胎盘(PFKFB3)和睾丸(PFKFB4)中表达。以往的研究主要集中在PFKFB3同工酶是肿瘤细胞中F2，6BP的主要来源，因为它的高激酶：磷酸酶比(~740：1)和在多种肿瘤中的过度表达。肿瘤细胞中高糖酵解率的特异性PFKFB同工酶的鉴定可能有助于开发新的药物，通过抑制糖酵解来抑制肿瘤生长。在初步研究中，我们用多重RT-PCR方法检测了20例肿瘤组织中四种PFKFB同工酶的mRNA表达，发现17/20的肿瘤组织中PFKFB4的mRNA(而不是PFKFB3)明显高于癌旁正常组织。我们推测，PFKFB4的高表达可能是由于不同的致癌途径汇聚在一起以增强糖酵解通量所致。我们发现，K-rasG12S+A549肺腺癌细胞的低氧暴露和H-rasG12V导入永生化的人支气管上皮细胞可增加PFKFB4的mRNA和蛋白表达。此外，细胞分离表明，PFKFB4蛋白而不是PFKFB3蛋白定位于细胞质，即PFK-1和糖酵解的细胞室。最后，瞬时siRNA沉默K-rasG12S+A549细胞中PFKFB4 mRNA的表达，降低了F2，6BP的稳态浓度，糖酵解通量和小鼠非锚定依赖性肿瘤生长。综上所述，这些初步研究为进一步研究PFKFB4同工酶作为抗肿瘤药物开发的新靶点提供了理论基础。我们推测，PFKFB4同工酶是转化细胞高糖酵解通量、存活、生长和扩散所必需的。我们计划通过以下具体目标来验证这一假说：1.检测PFKFB4合成的F2，6BP对糖酵解和线粒体代谢的需求。2.确定PFKFB4合成的F2，6BP对正常、永生化和ras转化的上皮细胞生长、侵袭和存活的需求。3.利用Cre-lox可诱导的小鼠基因敲除PFKFB4，检测PFKFB4基因组缺失对ras依赖肿瘤生长和代谢的影响。与公共卫生相关：我们预计shRNA沉默或PFKFB4同工酶的基因组缺失将减弱转化的上皮细胞的肿瘤潜能，相反，F2，6BP的增加(由于PFKFB4的过度表达)将增加糖酵解通量，促进肿瘤生长。这些发现可能验证了PFKFB4作为抑制糖酵解的新靶点。因此，通过计算筛选PFKFB4底物结合域的竞争性抑制剂来开发PFKFB4的小分子抑制剂来抑制癌症中的糖酵解通量，可能会降低与癌症相关的高死亡率。