Background: Overexpression of tissue plasminogen activator (t-PA) in pancreatic cancer cells promotes invasion and proliferation in vitro and tumour growth and angiogenesis in vivo. Aims: To understand the mechanisms by which t-PA favours cancer progression, we analysed the surface membrane proteins responsible for binding specifically t-PA and studied the contribution of this interaction to the t-PA promoted invasion of pancreatic cancer cells. Methods: The ability of t-PA to activate plasmin and a fluorogenic plasmin substrate was used to analyse the nature of the binding of active t-PA to cell surfaces. Specific binding was determined in two pancreatic cancer cell lines (SK-PC-1 and PANC-1), and complex formation analysed by co-immunoprecipitation experiments and co-immunolocalisation in tumours. The functional role of the interaction was studied in Matrigel invasion assays. Results: t-PA bound to PANC-1 and SK-PC-1 cells in a specific and saturable manner while maintaining its activity. This binding was competitively inhibited by specific peptides interfering with the interaction of t-PA with annexin II. The t-PA/annexin II interaction on pancreatic cancer cells was also supported by coimmunoprecipitation assays using anti-t-PA antibodies and, reciprocally, with antiannexin II antibodies. In addition, confocal microscopy showed t-PA and annexin II colocalisation in tumour tissues. Finally, disruption of the t-PA/annexin II interaction by a specific hexapeptide significantly decreased the invasive capacity of SK-PC-1 cells in vitro. Conclusion: t-PA specifically binds to annexin II on the extracellular membrane of pancreatic cancer cells where it activates local plasmin production and tumour cell invasion. These findings may be clinically relevant for future therapeutic strategies based on specific drugs that counteract the activity of t-PA or its receptor annexin II, or their interaction at the surface level.
Background: Tissue plasminogen activator (tPA) is the major activator of plasminogen in plasma. This serine protease is overexpressed by exocrine pancreas tumour cells, where it promotes tumour cell proliferation, growth, and invasion. Here we have explored the signalling pathways used by tPA to activate the proliferation of pancreatic cancer cells. Methods: Transcriptional profiling on cDNA micro arrays was used to analyse the pattern of gene expression in response to tPA compared to the response to epidermal growth factor (EGF) and platelet derived growth factor (PDGF). Results were confirmed using different biochemical assays in which specific kinase inhibitors or RNA interference were used. Results: Transcriptional profiling showed that tPA modulates the expression of a set of genes commonly regulated by EGF, but distinct from the major set of genes modulated by PDGF. This suggested that tPA and EGF share common signalling pathways, a conclusion supported by further experimental evidence. Firstly, we found that tPA induced a rapid and transient phosphorylation of the EGFR. Secondly, specific EGFR kinase inhibitors, but not PDGFR kinase inhibitors, abolished the tPA induced phosphorylation of the ERK1/2 kinases and cell proliferation. The mitogenic activity of tPA was also inhibited by siRNA depletion of EGFR, thus confirming the involvement of this receptor in tPA triggered signalling. Thirdly, we show that the signalling and mitogenic effects of tPA require its proteolytic activity, the activity of the metalloprotease-9 and active hb-EGF. Conclusion: Our results suggest that tPA induces proliferation by triggering a proteolytic cascade that sequentially activates plasmin, metalloprotease-9 (MMP-9) and hb-EGF. These events are required to activate the EGFR signalling pathway and cell proliferation.
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