Abstract
Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness, and resistance to treatment. Alpha-enolase (ENOA) is a key glycolytic enzyme that is also expressed on the surface of immune and tumor cells where it acts as a plasminogen receptor. In our laboratory it has been demonstrated that ENOA is expressed on the surface of several PDAC cell lines where is crucial for plasminogen-dependent invasion. A monoclonal antibody against ENOA inhibits this effect both in vitro and in vivo (unpublished data).
Aim: We investigated the effect of ENOA silencing in PDAC cell line in order to identify key proteins that are different modulated and clarify its multifunctional role in pancreatic tumorigenesis.
Methods: CF-PAC-1 human PDAC cell line was infected with a lentiviral vector delivering a short hairpin RNA (shRNA) targeting ENOA 3’UTR or a scrambled shRNA as a control. After silencing, ENOA mRNA was evaluated by real-time PCR and protein level was assessed by and Western Blot Analysis in each subcellular fraction (cytosolic, membrane and nuclear protein). Proliferation was monitored by cell counting and MTT assays. The cell cycle analysis was performed by cytofluorimetry. A pyruvate assay kit was used to determinate pyruvate production. The tumorigenicity of ENOA silenced cells was assessed by a colony forming assay and their invasive capability was tested by a transwell matrigel invasion assay. Adhesion capability of the cells was evaluated by plate fixing and cell counting. Finally the proteomes of ENOA silenced and control (scrambled or normal CF-PAC-1) cells were compared by LTQ-Orbitrap tandem mass spectrometry analysis and spectra count label-free quantification approach.
Results: After lentiviral infection, ENOA was silenced both at the transcript and at the protein level by around 80%, and the silencing was evident in all the cellular fractions. Compared to the control cells, ENOA silenced cells displayed a delay in proliferation and a decreased survival capability, even if the pyruvate production was not affected. This effect was due to a partially block of cell cycle in G2/M phase and not to an apoptotic events. In silenced cells, the capability to form colonies in soft agar was reduced to 40-50%, the invasion in response to plasminogen was abolished, and the adhesive capability decreased to 40%. Finally, more than 1500 proteins were identified from each sample by tandem mass spectrometry analysis and a large number of proteins were differentially expressed in ENOA silenced cells compared to control cells. These proteins were mainly involved in cell adhesion, cell metabolism and cell proliferation and mRNA analysis revealed that the most significantly downregulated proteins (cell-cell and cell-matrix adhesion regulators) were controlled either at the transcriptional or post-translational levels.
Conclusions: Together, this data confirm that ENOA in PDAC is not only a glycolytic enzyme, but is a key regulator of a broad spectrum of pathways involved in tumorigenesis. The mechanisms through which ENOA controls cell adhesion, proliferation and metabolism in PDAC are currently under investigation.
Citation Format: Moitza Principe, Michela Capello, Michelle Samuel Chattaragada, Weidong Zhou, Lance Liotta, Emanuel Petricoin, Francesco Novelli. Effects of alpha-enolase silencing in adenocarcinoma pancreatic cell line. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A65.
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