Targeting Glucose Metabolism in Cancer

描述（由申请人提供）：高糖酵解通量，即使在氧气存在的情况下，是肿瘤的中心代谢标志。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-脱氧葡萄糖正电子发射断层扫描（PET）证实，肿瘤比体内邻近正常组织多吸收10倍的葡萄糖。HIF-1a和myc的过度表达、ras的激活和p53功能的缺失通过激活一个由四个双功能6-磷酸果糖-2-激酶/果糖-2,6-双磷酸酶（PFKFB）组成的家族来刺激糖酵解，该家族合成果糖-2,6-二磷酸（F2,6BP），这是6-磷酸果糖-1-激酶（PFK-1）的变质激活剂，是糖酵解途径的重要控制点。据报道，PFKFB酶由四个基因（PFKFB1-4）编码，这些基因在不同的组织中表达：肝/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同工酶是肿瘤细胞中F2,6BP的主要来源，因为它的激酶：磷酸酶比例高（~740:1），并且在多种肿瘤中过表达。在癌细胞中高糖酵解率的特异性PFKFB同工酶的鉴定可能允许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4种PFKFB同工酶的mRNA表达，发现17/20的肿瘤中PFKFB4 mRNA（而不是PFKFB3）相对于邻近的正常组织显著升高。我们推测PFKFB4的高表达可能是由于不同的致癌途径聚集以增强糖酵解通量。我们发现低氧暴露于K-rasG12S+ A549肺腺癌细胞和将H-rasG12V导入永活的人支气管上皮细胞可增加PFKFB4 mRNA和蛋白的表达。此外，细胞分离显示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，短暂siRNA沉默K- rasG12S+ A549细胞中pfkfb4mrna的表达，降低了稳态F2浓度、6BP、糖酵解通量和不依赖锚定的小鼠肿瘤生长。综上所述，这些初步研究为进一步研究PFKFB4同工酶作为开发抗肿瘤药物的可能新靶点提供了依据。我们假设PFKFB4同工酶对于转化细胞的高糖酵解通量、存活、生长和传播是必需的。我们计划通过以下具体目标来检验这一假设：1。研究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-转化上皮细胞生长、侵袭性和存活的需求。3. 通过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。公共卫生相关性：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能，相反，F2,6BP的增加（来自PFKFB4的过表达）将增强糖酵解通量并促进肿瘤生长。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，通过开发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。

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.