Convergence and cooperativity between EGFR, TGFb, insulin and S100A8/A9 signaling in pancreatic ductal adenocarcinoma (PDAC)

Background. EGFR activation is required for TGFb1-induced expression of fibrotic genes. In PDAC, EGFR overexpression and TGFb signaling impairment by Smad4 inactivation are frequent. Signalling of other growth factors and inflammatory proteins, abundant in PDAC stroma, might also depend on EGFR activation.Objective: to verify whether a prolonged activation of the EGFR modifies TGFb1, insulin and S100A8/A9 signaling, in a Smad4 dependent/independent manner.Methods.BxPC3 (Smad4 HD) and Smad4 transfected BxPC3 (BxPC3-Smad4+) remained unstimulated (C) or were daily stimulated (S) for three days with EGF (100ng/mL). Ten minutes treatment with TGFb1(0.02ng/mL), insulin(50 mU) or S100A8/A9(10 nM) were followed by Reverse Phase Protein Array (RPPA) analysis of: MAPK,NF-kB,SRC/JAK/STAT3,PI3/AKT,inflammasome signaling pathways. RPPA data analysis consisted of calculating the concentration of each sample, after background correction and normalization. Intensities of stimulated cells were referred to controls and variations higher than 30% were considered. Results: in C-BxPC3-Smad4+, not in S- BxPC3-Smad4+, TGFb1 and insulin activated MAPK/ERK(pERK1/2;pMEK 1/2), SAPK/JNK(p-c-Jun;pSEK/MKK4) and p38 MAPK(pP38;TRAF2;pTAK1), while S100A8/A9 activated MAP/ERK signalling only. In S-BxPC3-Smad4+, these molecules induced MyD88 expression and NF-kB activation (pIKKa/b,pIRAK1,A20). In C-BxPC3, not in S-BxPC3, TGFb1 and S100A8/A9 inhibited NF-kB(IKKg,pIRAK1,A20) and PI3K/AKT(p-eNOS,pBAD,pAKT308,pAKT473,PI3K,p100a,PI3K,p85,pGSK3b) pathway. In S-BxPC3, S100A8/A9 induced HSP27, TRAF6 and BAD phosphorylation. RPPA results were confirmed by WB (pIkBa;pP38;pERK1/2;pAKT308;pAKT473).Conclusion. In Smad4-expressing cells, EGFR stimulation causes insulin, TGFb1 and S100A8/A9 signalling bypass from MAPK to NF-kB and MyD88, both implicated in favouring tumorigenesis. When Smad4 is HD, EGFR stimulation is permissive for the S100A8/A9 activation of HSP27 and TRAF6, both involved in favouring cell growth and invasion.