PANCREAS

Specific interaction of tissue-type plasminogen activator (t-PA) with annexin II on the membrane of pancreatic cancer cells activates plasminogen and promotes invasion in vitro

V M Díaz^1,*, M Hurtado², T M Thomson³, J Reventós², R Paciucci²

¹ Unitat de Recerca Biomèdica, Hospital Materno-Infantil, Hospitals Vall d’Hebrón, Pg Vall d’Hebrón 119-129, 08035 Barcelona, Spain, and Institut de Biologia Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, c Jordi Girona, 18-26, 08034, Barcelona, Spain
² Unitat de Recerca Biomèdica, Hospital Materno-Infantil, Hospitals Vall d’Hebrón, Pg Vall d’Hebrón 119-129, 08035 Barcelona, Spain
³ Institut de Biologia Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, c Jordi Girona, 18-26, 08034, Barcelona, Spain

Correspondence to:
Correspondence to:
Dr R Paciucci
Unitat de Recerca Biomèdica, URB pl 14 Hospital Materno-Infantil Vall d’Hebrón, Pg Vall d’Hebrón, 119-129, 08035 Barcelona, Spain; rpaciucci{at}vhebron.net

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 co-immunoprecipitation 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.

Abbreviations: t-PA, tissue-type plasminogen activator; rt-PA, recombinant t-PA; DFP-t-PA, t-PA inactivated by DFP; DFP, diisopropyl fluorophosphate; PAI-1, plasminogen activator inhibitor 1; u-PA, urokinase-type plasminogen activator; AMC, 7-amido-4-methylcoumarin; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; PBS, phosphate buffered saline; BSA, bovine serum albumin; rfu, relative fluorescence units

Keywords: tissue-type plasminogen activator; pancreatic cancer; annexin II; membrane receptor

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