Abstract
BackgroundWe have previously shown that in pancreatic ductal adenocarcinoma (PDA) cells, the glycolytic enzyme alpha-enolase (ENO1) also acts as a plasminogen receptor and promotes invasion and metastasis formation. Moreover, ENO1 silencing in PDA cells induces oxidative stress, senescence and profoundly modifies PDA cell metabolism. Although anti-ENO1 antibody inhibits PDA cell migration and invasion, little is known about the role of ENO1 in regulating cell-cell and cell-matrix contacts. We therefore investigated the effect of ENO1 silencing on the modulation of cell morphology, adhesion to matrix substrates, cell invasiveness, and metastatic ability.MethodsThe membrane and cytoskeleton modifications that occurred in ENO1-silenced (shENO1) PDA cells were investigated by a combination of confocal microscopy and atomic force microscopy (AFM). The effect of ENO1 silencing was then evaluated by phenotypic and functional experiments to identify the role of ENO1 in adhesion, migration, and invasion, as well as in senescence and apoptosis. The experimental results were then validated in a mouse model.ResultsWe observed a significant increase in the roughness of the cell membrane due to ENO1 silencing, a feature associated with an impaired ability to migrate and invade, along with a significant downregulation of proteins involved in cell-cell and cell-matrix adhesion, including alpha v/beta 3 integrin in shENO1 PDA cells. These changes impaired the ability of shENO1 cells to adhere to Collagen I and IV and Fibronectin and caused an increase in RGD-independent adhesion to vitronectin (VN) via urokinase plasminogen activator receptor (uPAR). Binding of uPAR to VN triggers integrin-mediated signals, which result in ERK1-2 and RAC activation, accumulation of ROS, and senescence. In shENO1 cancer cells, the use of an anti-uPAR antibody caused significant reduction of ROS production and senescence. Overall, a decrease of in vitro and in vivo cell migration and invasion of shENO1 PDA cells was observed.ConclusionThese data demonstrate that ENO1 promotes PDA survival, migration, and metastasis through cooperation with integrins and uPAR.
Highlights
We have previously shown that in pancreatic ductal adenocarcinoma (PDA) cells, the glycolytic enzyme alpha-enolase (ENO1) acts as a plasminogen receptor and promotes invasion and metastasis formation
Additional experiments performed with lower cell confluence and with images taken at the level of the apical surface showed that Control cell line (shCTRL) cells possess well-defined actin filaments, while short hairpin RNA targeting ENO1 3’UTR (shENO1) PDA cells display reduced adhesion to fibronectin and collagens and increased adhesion to vitronectin As we showed that the proteins involved in extracellular matrix (ECM) cell adhesion were downregulated in shENO1 cells, we evaluated the adhesive ability of shCTRL or shENO1 PDA cells on fibronectin (FN), collagen I (Col-I), collagen IV (ColIV), and vitronectin (VN)
ENO1 silencing had a major impact on the alpha v/beta 3 integrin, which accounts for the inability of ENO1-silenced cells to adhere to the ECM matrix and promote PDA invasion
Summary
We have previously shown that in pancreatic ductal adenocarcinoma (PDA) cells, the glycolytic enzyme alpha-enolase (ENO1) acts as a plasminogen receptor and promotes invasion and metastasis formation. In addition to its well-known enzymatic function during glycolysis, ENO1 acts as a plasminogen receptor on the cell surface [8, 10] promoting metastatic cancer invasion [11,12,13,14,15]. We employed biochemical and functional approaches to investigate molecules involved in cell adhesion and migration of ENO1-silenced (shENO1) PDA cells. As cell adhesion, survival and migration are dependent on integrin binding to the extracellular matrix (ECM), and subsequent signals, the roles of alpha V/beta 3 and alpha 5/beta 1 integrins, as well as uPAR (an ECM receptor) were evaluated in shENO1 PDA cells. Results from this study indicated that ENO1, by cooperating with integrins and uPAR, is a key regulator of cell survival, adhesion, and motility in PDA
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
