Integration of Glycolysis with the Epithelial-Mesenchymal Transition

项目概要 肿瘤细胞中锌指 E 盒结合同源盒 1 (ZEB1) 的过度表达可促进上皮细胞 间质转化（EMT）与增强的侵袭和转移能力以及 获得癌症干细胞特征。了解 ZEB1 的监管方式应该允许 鉴定可用于治疗过度转移性癌症的药物靶标 表达ZEB1，包括胰腺癌和非小细胞肺癌（NSCLC）。 HIF-1¿-诱导型 6- 磷酸果糖-2-激酶/果糖-2,6-双磷酸-3 (PFKFB3) 在肿瘤中过度表达，激活 通过其产物果糖-2,6-二磷酸进行糖酵解，最近发现它是细胞周期蛋白的激活剂 依赖性激酶（Cdks）。鉴于越来越多的证据表明细胞周期调节剂的作用，例如 对于 EMT 中的 Cdks，我们假设 PFKFB3 可能对于获取/维护 肿瘤细胞的间质特征。在初步研究中，我们证明 A549 中的 PFKFB3 沉默 NSCLC细胞和MIA PaCa-2胰腺癌细胞导致ZEB1表达显着下降 并逆转 EMT（通过 E-钙粘蛋白（上皮标记物）增加和减少来评估） 波形蛋白/纤连蛋白（间充质标记物）水平。我们进一步表明 PFKFB3 的敲低降低 视网膜母细胞瘤 (RB) 蛋白（Cdk 靶标）的磷酸化以及 STAT3 蛋白的表达， 最近发现这两者都可以调节ZEB1的表达。因此我们假设 肿瘤细胞中 PFKFB3 的上调通过 RB 和/或 STAT3 依赖性增加 ZEB1 表达 机制，导致获得/维持与增加相关的间充质表型 转移能力和干性。为了检验这一假设，我们将追求以下具体目标：（1）研究 PFKFB3 与 ZEB1 的功能相互作用及其对侵袭和癌症干细胞特性的影响 体外； (2) 检查 PFKFB3 与 ZEB1 在转移性进展中的功能相互作用 体内胰腺腺癌。在这些研究结束时，我们期望建立一个新的功能 PFKFB3 对 ZEB1 基因和 EMT 的调节，这可能对 治疗侵袭性、转移性癌症，例如胰腺癌。

PROJECT SUMMARY Overexpression of Zinc finger E-box binding homeobox 1 (ZEB1) in tumor cells promotes the epithelial- mesenchymal transition (EMT) that is associated with enhanced invasive and metastatic capabilities and the acquisition of cancer-stem cell characteristics. Understanding how ZEB1 is regulated should permit the identification of druggable targets that can be exploited for the treatment of metastatic cancers that over- express ZEB1, including pancreatic and non-small cell lung cancers (NSCLC). The HIF-1¿-inducible 6- phosphofructo-2-kinase/fructose-2,6-bisphosphase-3 (PFKFB3) is over-expressed in tumors, activates glycolysis via its product fructose-2,6-bisphosphate and was recently found to be an activator of cyclin- dependent kinases (Cdks). Given the accumulating evidence suggesting a role for cell cycle regulators such as the Cdks in the EMT, we postulated that PFKFB3 may be essential for the acquisition/maintenance of mesenchymal traits in tumor cells. In preliminary studies, we demonstrate that PFKFB3 silencing in A549 NSCLC cells and MIA PaCa-2 pancreatic cancer cells causes a marked decrease in ZEB1 expression and reverses the EMT, as assessed by increased E-cadherin (an epithelial marker) and decreased vimentin/fibronectin (mesenchymal markers) levels. We further show that knockdown of PFKFB3 decreases the phosphorylation of the retinoblastoma (RB) protein, a Cdk target, and the expression of the STAT3 protein, both of which have recently been found to regulate ZEB1 expression. We therefore hypothesize that upregulation of PFKFB3 in tumor cells increases ZEB1 expression via an RB and/or STAT3-dependent mechanism, leading to the acquisition/maintenance of a mesenchymal phenotype associated with increased metastatic ability and stemness. The following specific aims will be pursued to test this hypothesis: (1) To study the functional interaction of PFKFB3 with ZEB1 and its effect on invasion and cancer stem cell properties in vitro; and (2) To examine the functional interaction of PFKFB3 with ZEB1 in the progression of metastatic pancreatic adenocarcinoma in vivo. At the end of these studies, we expect to have established a novel function for PFKFB3 in the regulation of the ZEB1 gene and EMT, which may have therapeutic implications for the treatment of aggressive, metastatic cancers such as pancreatic adenocarcinoma.