The role of Smad4/TGFB signaling in tumorigenesis of pancreatic ductal adenocarcinoma (PDAC).

Abstract

748 Background: Pancreatic cancer ranks 4th among cancers as a cause of death in the United States and 85- 90% of pancreatic cancer are pancreatic ductal adenocarcinoma (PDAC). Previous studies have demonstrated 2 major types of precursors to PDAC including pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN); the mechanisms by which IPMNs give rise to PDAC are incompletely defined. We hypothesize that SMAD4 signaling is specifically involved in IPMN pathway with PanINs and IPMN having partially overlapping signaling pathways and molecular markers. Methods: We performed immunohistochemistry staining on different precursor lesions of mouse pancreatic tissue (PanIN precursor: Ptf1aCre LSL-KrasG12D; IPMN precursor: Ptf1aCreERT LSL- KrasG12D Smad4fl/fl treated with tamoxifen) using markers of different IPMN subtypes and signaling pathways known to be involved in PDAC including, MUC1, MUC2, MUC5AC, SMAD4, ERK1/2, SOX9, and YAP. We treated IPMN cells lines derived from Ptf1aCreERTM LSL-KrasG12D GnasR201C mice with TGF-β, BMP-2, or vehicle control and quantified RNA expression of Smad7, Serpine1, Id2, and normalized to Pmm1 expression. We performed a western blot to detect the protein expression of phospho-SMAD1, phospho-SMAD2, SMAD1, and SMAD2. Results: Both PanIN and IPMN mouse models exhibited strong expression of MUC5AC and MUC1, reflecting the pancreatobiliary subtype. The tumor cells in both models expressed phosphorylated substrates of protein kinase A, phosphorylated ERK1/2, and SOX9. Regional differences were noted for SOX9 signaling with IPMN demonstrating a greater expression around the outer edges of the lesion, compared to the center. PanIN model demonstrated a more prominent nuclear YAP staining. Treatment of BMP2 and TGF-β in pancreatic IPMN cells from Ptf1aCreERTM LSL-KrasG12D GnasR201C cell line significantly increased RNA levels of Serpine1 (2.4 and 199.6, respectively; n = 4; p < 0.05) and Smad7 (2.9 and 6.3, respectively; n = 4; p < 0.05). BMP2 treatment significantly increased RNA levels of Id2 (1.9; n = 4; p < 0.05) while TGF-β significantly decreased Id2 level (0.3; n = 4; p < 0.05). Phosphorylated-SMAD2 was detected only with TGF-β treatment while phosphorylated-SMAD1 was detected with vehicle, TGF-β, and BMP2 treatment with the highest level with BMP2 treatment. Conclusions: Knocking out Smad4, concurrent with activating KrasG12D mutation, induces a specific IPMN progression model that shares similar molecular markers as the PanIN progression model, shown in Kras mutation alone. SMAD4 signaling remains intact in IPMN lesions that arise from activating the KrasG12D GnasR201C mutation, possibly suggesting that Smad4 mutation induces the IPMN-PDAC progression model that differs from Gas-cAMP-PAK-YAP1 cascade found in GNAS signaling.