Epidermal growth factor mediates detachment from and invasion through collagen I and Matrigel in Capan-1 pancreatic cancer cells

Abstract

Background: Pancreatic adenocarcinoma is a highly invasive neoplasm. Epidermal growth factor (EGF) and its receptor are over expressed in pancreatic cancer, and expression correlates with invasion and metastasis. We hypothesized that EGF receptor and integrin signalling pathways interact in mediating cellular adhesion and invasion in pancreatic cancer, and that invasiveness correlates temporally with detachment from extracellular matrix.

Methods: We tested this hypothesis by investigating the role of EGF in mediating adhesion to and invasion through collagen I and Matrigel in the metastatic pancreatic adenocarcinoma cell line Capan-1. Adhesion and invasion were measured using in vitro assays of fluorescently-labeled cells. Adhesion and invasion assays were also performed in the primary pancreatic adenocarcinoma cell line MIA PaCa-2.

Results: EGF inhibited adhesion to collagen I and Matrigel in Capan-1 cells. The loss of adhesion was reversed by AG825, an inhibitor of erbB2 receptor signalling and by wortmannin, a PI3K inhibitor, but not by the protein synthesis inhibitor cycloheximide. EGF stimulated invasion through collagen I and Matrigel at concentrations and time courses similar to those mediating detachment from these extracellular matrix components. Adhesion to collagen I was different in MIA PaCa-2 cells, with no significant change elicited following EGF treatment, whereas treatment with the EGF family member heregulin-alpha elicited a marked increase in adhesion. Invasion through Matrigel in response to EGF, however, was similar to that observed in Capan-1 cells.

Conclusion: An inverse relationship exists between adhesion and invasion capabilities in Capan-1 cells but not in MIA PaCa-2 cells. EGF receptor signalling involving the erbB2 and PI3K pathways plays a role in mediating these events in Capan-1 cells.

Figure 1

Adhesion of cells to various extracellular matrix components and the effect of EGF. Adhesion was tested under conditions of no growth factor (open symbols) and treatment with 10 nM EGF (closed symbols) for 8 hours. Results are expressed as % adhesion ± SEM from three separate experiments, each performed in triplicate wells. * p < 0.003; ** p < 0.05.

Figure 2

Effects of various inhibitors on adhesion of cells to collagen I. Adhesion assays on collagen I were performed with no growth factor (open bars), 10 nM EGF (closed bars) or 10 nM HRG-α (hatched bars). Shown are the results of three separate experiments, each performed in triplicate. * p < 0.001. **p < 0.02.

Figure 3

Effects of cycloheximide and AG825 on adhesion of MIA PaCa-2 cells to collagen I. Adhesion assays on collagen I using were performed with no growth factor (open bars), 10 nM EGF (closed bars) or 10 nM HRG-α (hatched bars). Shown are the results of three separate experiments, each performed in triplicate. * p < 0.001.

Figure 4

Effects of EGF and HRG-α on invasion through Matrigel in Capan-1 and MIA PaCa-2 cells. Cells were cultured on invasion chambers coated with Matrigel, and invasion measured following treatment with no growth factor, 10 nM EGF, or 10 nM HRG-α. Shown are the results of 3 experiments, each performed in triplicate wells. *p < 0.05 between the no growth factor group and the EGF treatment group for each cell type.

Figure 5

Time course of EGF-mediated inhibition of adhesion. The adhesion assay was performed and the experiment terminated at various time points. Adhesion was measured without and with EGF treatment. Shown are the results of one experiment performed in triplicate wells, which was repeated twice with similar results.

Figure 6

The time course of EGF-mediated invasion through Matrigel. Cells were labeled and invasion through chambers performed in the absence or presence of 10 nM EGF. Shown are the results of one experiment performed in triplicate wells, which was repeated twice with similar results.

Figure 7

Dose response of EGF-induced detachment from collagen I and stimulation of invasion through Matrigel. Adhesion and invasion were measured following incubation with escalating doses of EGF. Shown are the results of three experiments, each performed in triplicate.

Figure 8

Expression of EGFR, α₂/β₁ integrin in Capan-1 cells. Cells were cultured on Transwell inserts, and confocal immunofluorescence microscopy performed following incubation with primary antibodies against EGF-R (A), α₂ integrin (B) and β₁ integrin (C). Negative controls with primary antibody omitted did not show a detectable signal.

Figure 9

Phalloidin-TRITC staining of cells with and without EGF treatment. Cells were cultured on Transwell inserts, and confocal immunofluorescence microscopy performed following incubation with TRITC-conjugated phalloidin, in the absence (A) or presence (B) of 10 nM EGF. Arrow shows focal area of actin staining consistent with a focal adhesion complex.

Figure 10

Soluble EGF levels in incubation media from Capan-1 cells compared to media from human gallbladder myofibroblasts. Cells were cultured in media with or without 10% FBS. Shown are the results of one experiment, which was repeated with similar results (A). B. Confocal immunofluorescence image of confluent Capan-1 cells stained for EGF.