Loss of Trefoil Factor 2 From Pancreatic Duct Glands Promotes Formation of Intraductal Papillary Mucinous Neoplasms in Mice

Abstract

Background & Aims

Little is known about the origin of pancreatic intraductal papillary mucinous neoplasms (IPMN). Pancreatic duct glands (PDGs) are gland-like outpouches budding off the main pancreatic ducts that function as progenitor niche for the ductal epithelium; they express gastric mucins and have characteristics of side-branch IPMN. We investigated whether PDGs are a precursor compartment for IPMN and the role of trefoil factor family 2 (TFF2)— a protein expressed by PDGs and the gastric mucosa that are involved in epithelial repair and tumor suppression.

Methods

We obtained pancreatectomy specimens from 20 patients with chronic pancreatitis, 13 with low-grade side-branch IPMN, and 15 patients with PDAC; histologically normal pancreata were used as controls (n=18). Samples were analyzed by immunohistochemistry to detect TFF1 and TFF2 and cell proliferation. We performed mitochondrial DNA mutational mapping studies to determine the cell lineage and fate of PDG cells. Pdx1-Cre;LSL-KRAS ^G12D (KC) mice were bred with TFF2-knockout mice to generate KC/ Tff2−/− and +/− mice. Pancreata were collected and histologically analyzed for formation of IPMN, pancreatic intraepithelial neoplasias, and PDAC, in addition to proliferation and protein expression. Human pancreatic ductal epithelial cells and PDAC cell lines were transfected with vectors to overexpress or knock down TFF2 or SMAD4.

Results

Histologic analysis of human samples revealed gastric-type IPMN to comprise 2 molecularly distinct layers: a basal crypt segment that expressed TFF2 and overlying papillary projections. Proliferation occurred predominantly in the PDG-containing basal segments. Mitochondrial mutation mapping revealed a 97% match between the profiles of proliferating PDG cells and their overlying non-proliferative IPMN cells. In contrast to KC mice, 2-month-old KC/ Tff2+/− and KC/ Tff2−/− mice developed prominent papillary structures in the duct epithelium with cystic metaplasia of the PDG, which resembled human IPMN; these expressed gastric mucins (MUC5AC and MUC6) but not the intestinal mucin MUC2. KC/TFF2-knockout mice developed a greater number and higher grade of pancreatic intraepithelial neoplasias than KC mice, and 1 mouse developed an invasive adenocarcinoma. Expression of TFF2 reduced proliferation of PDAC cells 3-fold; this effect required upregulation and activation of SMAD4. We found expression of TFF2 to be downregulated in human PDAC by hypermethylation of its promoter.

Conclusions

In histologic analyses of human IPMNs, we found PDGs to form the basal segment and possibly serve as a progenitor compartment. TFF2 has tumor suppressor activity in the mouse pancreas and prevents formation of mucinous neoplasms.