S100A8 and S100A9 Target Akt, mTOR and NF-kB Signalling in Pancreatic Cancer Cells
Context S100A8/S100A9 inflammatory proteins are suggested to be involved in pancreatic cancer (PaCa) progression. S100A8/A9 expression in the neoplastic pancreas correlate with SMAD4 mutational status suggesting possible interactions with TGF-b1. Objective To ascertain whether S100A8/S100A9 differently affect Akt, mTOR and NF-kB signalling in PaCa cells with different aggressiveness and whether these molecules interact with TGF-b1. Methods Western blotting analyses were used to assess the effects of S100A8, S100A9 and S100A8/A9 on Akt (Ser473, Thr308), mTOR (Ser2448) and NF-kB (p-IkB-a) in BxPC3, Capan1 and MiaPaCa2 PaCa cells lines. S100A8, S100A9, S100A8/A9 were incubated with equimolar concentrations of calcium and TGF-b1 for 24 h at 37°C. Following MALDI-TOF-MS analyses were performed. Results In BxPC3 Akt Thr308 was phosphorylated by S100A8/A9, while in Capan1 and MiaPaCa2 S100A8/A9, S100A8 and S100A9 phosphorylated both Akt sites. In Capan1 and MiaPaCa2, not in BxPC3, S100A8, S100A9 and S100A8/A9 caused significant Ser2448 phosphorylation. S6RP, downstream effector of mTORC1, was phosphorylated (Ser235/236) only in S100A8 treated MiaPaCa2. A strong NF-kB activation was induced by S100A8 in BxPC3, by S100A9 and S100A8/A9 in Capan1. NF-kB was inhibited by S100A8 and S100A8/A9, while it was activated by S100A9 in MiaPaCa2. In the presence of TGF-b1 S100A8/A9 effects on Akt Thr308 and Ser473 phosphorylation were significantly modified, suggesting the existence of interactions between TGF-b1 and S100A8/A9. In the presence of calcium ions S100A8 and S100A9 formed, as expected, homo- (21663 m/z and 28346 m/z) and hetero- (25153 m/z) dimers (MALDI-TOF-MS). An absolute new finding was the identification of hetero-complexes formed by S100A9 and TGF-b1 (39803 m/z). Conclusion S100A8/A9 proteins in pancreatic cancer might favour cancer cell growth by inducing Akt, mTOR and NF-kB. In the less invasive BxPC3 cells S100A8 activates NF-kB. In more aggressive Capan1 and MiaPaCa2 cells S100A8, S100A9 and S100A8/A9 activate mainly Akt and mTORC1, not NF-kB pathways. TGF-b1 was demonstrated to be a new binding partner of S100A9 and this will open new fields of investigation on these interesting and complex inflammatory proteins in the PaCa setting.
Copyright (c) 2014 Stefania Moz, Dania Bozzato, Andrea Padoan, Carlo Federico Zambon, Paola Fogar, Cosimo Sperti, Eliana Greco, Filippo Navaglia, Michela Pelloso, Elisa Rossi, Claudio Pasquali, Sergio Pedrazzoli, Mario Plebani, Daniela Basso
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