The inflammatory calcium binding protein S100A8 and its N-terminal proteolytic fragment interact with transforming growth factor-beta1 (TGF-B1) and alter AKT, mTOR and NF-kB cancer cell signalling

Introduction: in pancreatic cancer S100A8 is highly expressed by stromal cells when SMAD 4 is not mutated or by cancer cells when SMAD 4 is mutated, suggesting a link between TGF -b1 and S100A8 pathways. The proteolytic fragment of S100A8, NT-S100A8, highly abundant in pancreatic cancer, is involved in altering insulin secretion and action. Aim: to ascertain whether S100A8 and NT-S100A8 interacts with TGF -b1 in altering intracellular calcium, NF -kB, Akt and mTOR signalling. Methods: BxPC3 cells were stimulated with S100A8 (10nM), NT-S100A8 (50nM) alone or combined with TGF -b1 (0.02ng/mL). Intracellular calcium was monitored by Fluo4 (epifluroescence). Akt (Ser473,Thr308), mTOR (Ser2448), NF -kB (p-IkB-a) were WB analyzed. Results: NT-S100A8 evoked a train of intracellular calcium fluxes after 150 seconds lag time, which was reduced to few seconds in the presence of TGF -b1. S100A8 or TGF -b1 alone did not alter intracellular calcium. NF -kB signalling was activated in a calcium-dependent manner by S100A8 and by NT-S100A8 in the presence of TGF -b1. Akt Ser473 phosphorylation was reduced by NT-S100A8, TGF -b1 but mainly by their combination. AktThr308 was not affected by the studied molecules. mTOR phosphorylation (Ser2448) was induced by S100A8 and, at a lesser degree, by TGF -b1 and NT-S100A8. The phosphorylation (Ser235/236) of the downstream effector of mTORC1, S6RB, was reduced by TGF -b1 and NT-S100A8 independently, not by S100A8. Conclusions: NT-S100A8 mimics TGF -b1 inhibitory effects on Akt and mTOR signalling. These two molecules co-operate in inhibiting Akt probably by altering intracellular calcium, while they co-operate in activating NF -kB in a calciumindependent manner mimicking the entire S100A8 molecule effect.