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）：即使在有氧存在的情况下，高糖酵解通量也是肿瘤性肿瘤的核心代谢标志。癌细胞优先利用糖酵解来满足其增加的能量和生物合成需求。这种代谢表型通过 2-[18F]-氟-2-脱氧-葡萄糖的正电子发射断层扫描 (PET) 得到证实，这表明肿瘤在体内吸收的葡萄糖比邻近正常组织多 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-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶 (PFKFB) 家族来刺激糖酵解，该家族合成果糖-2,6-二磷酸 (F2,6BP)，这是一种 6-磷酸果糖-1- 的变构激活剂 激酶（PFK-1）是糖酵解途径中的重要控制点。 PFKFB 酶由四种基因 (PFKFB1-4) 编码，据报道在不同组织中表达：肝脏/骨骼肌 (PFKFB1)、心脏 (PFKFB2)、胎盘 (PFKFB3) 和睾丸 (PFKFB4)。先前的研究主要集中在 PFKFB3 同工酶作为肿瘤细胞中 F2,6BP 的主要来源，因为它具有较高的激酶：磷酸酶比率（约 740:1）并且在多种肿瘤中过度表达。鉴定导致癌细胞中高糖酵解率的特定 PFKFB 同工酶可能有助于开发通过抑制糖酵解来抑制肿瘤生长的新型药物。在初步研究中，我们通过多重 RT-PCR 测量了 20 个肿瘤组织中四种 PFKFB 同工酶的 mRNA 表达，发现 17/20 肿瘤中的 PFKFB4 mRNA（而不是 PFKFB3）相对于邻近的正常组织显着增加。我们推测 PFKFB4 的高表达可能是由不同的致癌途径汇聚以增强糖酵解通量引起的。我们发现，K-rasG12S+ A549 肺腺癌细胞的缺氧暴露以及将 H-rasG12V 引入永生化人支气管上皮细胞中会增加 PFKFB4 mRNA 和蛋白的表达。此外，细胞分级显示，PFKFB4 蛋白（而非 PFKFB3）定位于细胞质，即 PFK-1 和糖酵解的细胞区室。最后，K-rasG12S+ A549 细胞中 PFKFB4 mRNA 表达的瞬时 siRNA 沉默降低了小鼠中 F2,6BP 的稳态浓度、糖酵解通量和贴壁依赖性肿瘤生长。总而言之，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可能新靶点提供了理论基础。我们假设 PFKFB4 同工酶是转化细胞的高糖酵解通量、存活、生长和扩散所必需的。我们计划通过实现以下具体目标来检验这一假设： 1. 检查 PFKFB4 合成的糖酵解和线粒体代谢对 F2,6BP 的需求。 2.确定正常、永生化和ras转化上皮细胞的生长、侵袭和存活对PFKFB4合成的F2,6BP的需求。 3. 使用 Cre-lox 诱导小鼠敲除 PFKFB4，研究 PFKFB4 基因组缺失对体内 ras 依赖性肿瘤生长和代谢的影响。公共健康相关性：我们预计 shRNA 沉默或 PFKFB4 同工酶的基因组缺失将减弱转化上皮细胞的肿瘤潜力，相反，F2,6BP 的增加（来自 PFKFB4 的过度表达）将增强糖酵解通量并促进肿瘤生长。这些发现可能证实 PFKFB4 作为抑制糖酵解的新靶点。因此，通过计算筛选 PFKFB4 底物结合结构域的竞争性抑制剂来开发 PFKFB4 小分子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。