SMAD4 Deletion and EGF Co-Operate in Favoring mTOR Activation in PDAC Cells

Abstract

Context EGFR overexpression occurs early, while loss of SMAD4 occurs in more advanced PDAC. Loss of SMAD4 alters TGFb1 signalling, associates with a reduced stromal and an increased cancer cell expression of S100A8/A9. Objective To ascertain whether the effects on NF-kB, Akt, mTOR and MAPK signalling pathways exerted by S100A8/A9, TGFb1 and EGF depend on SMAD4 deletion and whether they are sensitive to EGF chronic exposure. Methods BxPC3 (SMAD4 homozygous deletion) and SMAD4 expressing BxPC3 (BxPC3-SMAD4+; plasmid expression vector) were stimulated with EGF (100 ng/mL), S100A8/A9 (10 nM) and TGFb1 (0.02 ng/mL) alone or combined. Cells were both left untreated or were pre-treated with EGF for 3 days. Total protein lysates were used for the WB analysis of: Akt (Ser473, Thr308), mTOR (Ser2448, Ser2481), NF-kB (p-IkB-a), MAPK (p-p38, pErk 1/2). Results In BxPC3-SMAD4+ cells, EGF activated, while TGFb1 and S100A8/A9 inhibited Akt and MAPK. In these cells, S100A8/A9 and EGF stimulated, while TGFb1 inhibited NF-kB. SMAD4 deletion did not affect EGF signalling, reverted TGFb1 and S100A8/A9 effects on Akt, and allowed mTOR activation after TGFb1, S100A8/A9 and EGF treatments. EGF pre-treatment of BxPC3-SMAD4+ caused de-sensitization of NF-kB and MAPK to all stimuli, which inhibited Akt and activated mTOR. In the same conditions loss of SMAD4 caused NF-kB, MAPK and mTOR response to all stimuli. Only in BxPC3-SMAD4+ S100A8/A9 synergized with TGFb1 in inhibiting Akt. Conclusion SMAD4 deletion in pancreatic cancer cell and chronic treatment with EGF co-operate in activating pro-proliferative and pro-metastatic pathways when cells are treated with growth factors, inflammatory proteins and TGFb1.