Epidermal growth factor (EGF) chronic stimulation causes Smad4- dependent signaling bypass in pancreatic ductal adenocarcinoma (PDAC)

Context EGF and EGFR overexpression is an early, while Smad4 inactivation is a late event in PDAC progression. Acquired resistance to EGFR-targeted therapies might depend on the activation of bypass signaling pathways. Objective To verify whether a prolunged exposure of PDAC cells to EGF activates bypass signaling and whether this event is Sma4 dependent. Methods BxPC3 Smad4 Homozygous deletion (HD) and Smad4-transfected BxPC3 (BxPC3-Smad4+) remained unstimulated (C) or were daily stimulated (S) for three days with EGF (100ng/mL). Then, EGF treatment (100 ng/mL for 10 minutes) was followed by cell collection for Reverse Phase Protein Array (RPPA) analysis of: MAPK, NF-kB, SRC/JAK/STAT3, PI3K/AKT, inflammasome signaling pathways. RPPA data analysis consisted of calculating the concentration of each sample, after background correction and normalization (signal intensities gained from a image software). Intensities of stimulated cells were referred to controls and variations higher than 30% were considered. Results In C-BxPC3, EGF activated MAPK/ERK (pERK1/2) only. In C-BxPC3-Smad4+, EGF activated SRC/JAK/STAT3 (pc-SRC;pRIP2;pJAK2), MAPK/ERK (pERK1/2;pMEK 1/2) and SAPK/JNK (pc-Jun;pSEK/MKK4), not p38 MAPK (pP38;TRAF2;pTAK1). In S-BxPC3 and in S-BxPC3-Smad4+, EGF did not activate any MAPK pathway not SRC/JAK/STAT3. In S-BxPC3-Smad4+, EGF activated NF-KB (pIkBa;IKKg;pIRAK1;A20) and inhibited the inflammasome (pSTAT4;ACS;Caspase1;MyD88), while in S-BxPC3 EGF inhibited only the inflammasome pathway. The PI3K/AKT pathway was never affected by EGF. RPPA results were confirmed by western blot (WB) (pP38;pERK1/2;pAKT308;pAKT473). Conclusion EGRF chronic stimulation causes Smad4-dependent signaling bypass from MAPK to NF-KB in Smad4-expressing cells. Smad4 HD renders PDAC cells low responsive to EGF acute or chronic stimulation. These findings might explain the PDAC-low response rate to EGRF-targeted therapies.