Integration of Glycolysis with the Epithelial-Mesenchymal Transition

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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.