Trefoil factor 1 stimulates both pancreatic cancer and stellate cells and increases metastasis

Abstract

Objectives: Trefoil factor 1 (TFF1) is a stable secretory protein expressed widely in the gastrointestinal mucosa that is also expressed in pancreatic ductal adenocarcinoma (PDAC). In the current study, we documented the extent and timing of TFF1 expression and investigated the effects of TFF1 on PDAC cells and stellate cells, the primary cells of the PDAC stroma.

Methods: Trefoil factor 1 expression in pancreatic cancer tissues and cell lines was analyzed using microarray, quantitative reverse transcriptase-polymerase chain reaction, and immunohistochemistry. The effects of recombinant TFF1 on cell growth, migration, and invasion of pancreatic cancer cell lines and immortalized human pancreatic stellate cells (HPSCs) were analyzed using MTS and Matrigel-coated invasion chambers. In vivo studies were also conducted in which Mpanc-96 cells stably expressing TFF1 were implanted orthotopically into nude mice.

Results: Trefoil factor 1 was highly increased in preneoplastic lesions. Recombinant TFF1 stimulated motility of both cancer and HPSCs. In contrast, only HPSC cell growth was increased by TFF1. In vivo studies showed that overexpression of TFF1 in PDAC cells did not affect primary tumor growth but greatly increased metastasis.

Conclusions: The present data demonstrate that TFF1 influences both PDAC cells and stellate cells and stimulates metastasis.

FIGURE 1

Trefoil factor 1 expression in pancreatic cancer. A, Quantitative RT-PCR showing the expression of TFF1 mRNA in pancreatic cancer tissue using Β-actin as control. Insert shows representative RT-PCR. B, Reverse transcriptase–polymerase chain reaction showing the expression of TFF1 in pancreatic cancer cell lines and HPDE and in HPSC cells using Β-actin as control. C, Immunohistochemistry showing the absence of TFF1 in normal and chronic pancreatitis tissues and early expression of TFF1 in PanINs^– and also in pancreatic adenocarcinoma.

FIGURE 2

Exogenous TFF1 stimulates pancreatic cancer cell invasion but not proliferation in vitro. A, Trefoil factor 1 stimulated pancreatic cancer cell invasion in a concentration-dependent manner. Wild-type Mpanc-96 cells (20,000 cells) were added into BioCoat Matrigel invasion upper chamber, and different concentrations of TFF1 (0–100 nM) were added into the lower chamber. After 24 hours, noninvaded cells on the upper membrane were removed, and cells invading into the lower membrane were quantified photometrically using MTS reagent added 1 hour before taking the reading. Dulbecco’s modified Eagle medium containing 10% or 0% serum was used as control. B, Photographs of representative membranes of migrated Mpanc-96, BxPc3, and Panc-1 after Diff-Quick staining are shown. C, Trefoil factor 1 did not stimulate pancreatic cancer cell proliferation. Wild-type Mpanc-96 and BxPc-3 cells (5000 cells) were plated on 96-well plates, and TFF1 (0 and 100 nM) was added to the cells for 48 hours. Cell numbers were estimated by MTS assay. Data shown are mean ± SEM for 3 experiments. *P < 0.05 versus control.

FIGURE 3

Exogenous TFF1 stimulates PSC (HPSC) migration and proliferation in vitro. A, Trefoil factor 1 stimulated PSC migration in a concentration-dependent manner. Stellate cells (20,000 cells) were added into BioCoat migration upper chamber, and different concentrations of TFF1 (0–100 nM) were added into the lower chamber. After 8 hours, nonmigrated cells in the upper membrane were removed, and cells migrated into the lower membrane were quantified photometrically using MTS reagent added 1 hour before taking the reading. Dulbecco’s modified Eagle medium containing 10% or 0% serum was used as control. B, Photographs of representative membranes after Diff-Quick staining are also shown. C, Trefoil factor 1 stimulated PSC proliferation in a concentration-dependent manner. Stellate cells (5000 cells) were plated on 96-well plates, and TFF1 (0–100 nM) was added to the cells for 48 hours. Cell numbers were estimated by MTS assay. Data shown are mean ± SEM for 3 experiments. *P < 0.05 versus control.

FIGURE 4

Trefoil factor 1 increases total tumor burden in vivo. A, The growth of tumors formed from Mpanc-96 cells stably transfected with control or TFF1-expressing vectors was analyzed in 4-week-old male athymic nude mice. Both control and TFF1-expressing cells were stably transfected with luciferase gene, and 1 × 10⁶ cells were injected into the pancreas. Bioluminescent imaging was used to estimate overall tumor burdens from 6 animals in each group for 6 weeks. B, The animals were killed, and tumor weight was measured at the end of the experiment. *P < 0.05 versus control.

FIGURE 5

Trefoil factor 1 increases metastasis in vivo. The metastasis from Mpanc-96 cells stably transfected with control or TFF1-expressing vectors were analyzed in 4-week-old male athymic nude mice. Both control and TFF1-expressing cells were stably transfected with luciferase gene, and 1 × 10⁶ cells were injected into the pancreas. Bioluminescent imaging was used to estimate metastasis from 6 animals in each group for 6 weeks. A, Trefoil factor 1 expression induces invasion as early as second week. B–D, Trefoil factor 1 expression increased metastasis to peritoneal cavity, liver, and lung, measured at the end of the experiment. Columns, mean for 6 animals (*P < 0.05); bars, SEM.