The alpha2beta1 integrin mediates the malignant phenotype on type I collagen in pancreatic cancer cell lines

Abstract

Pancreatic cancer is characterised by a hallmark desmoplastic response that includes upregulated expression of the extracellular matrix, and type I collagen in particular. Recent studies indicate that pancreatic cancer cells stimulate type I collagen synthesis in adjacent stellate cells, and that this upregulated type I collagen expression promotes the malignant phenotype in tumour cells as defined by increased proliferation, resistance to chemically induced apoptosis, and increased tumorigenesis. The integrin specificity of this interaction between type I collagen and tumour cells was not identified, however. In the present study, we examined eight pancreatic cancer cell lines for adhesion, proliferation, and migration, on types I and IV collagen, fibronectin, laminin, and vitronectin, as well as integrin expression. Our results indicate, for the overwhelming majority of cell lines, that type I collagen promotes the strongest adhesion, proliferation, and migration relative to the other substrates tested. Utilising function-blocking monoclonal antibodies directed against particular integrin subunits in cell adhesion and migration inhibition assays, we demonstrate further that the malignant phenotype on type I collagen is mediated specifically by the alpha2beta1 integrin. These results identify alpha2beta1 integrin-mediated adhesion to type I collagen as a potential therapeutic target in the treatment of pancreatic cancer.

Figure 1

Type I collagen promotes maximal adhesion of pancreatic cancer cell lines compared to other ECM proteins. Cell adhesion assays were conducted as described in Materials and Methods with Capan-1, CFPAC, Colo-357, FG, AsPC-1, BxPC-3, MiaPaCa-2, and Panc-1 cells. CI, □ – type I collagen; CIV, Δ – type IV collagen; Fn, ▴ – fibronectin; Ln, • – laminin; and Vn, ▪ – vitronectin. Results are expressed as the mean±s.e.m. from two independent experiments conducted in duplicate. Nonspecific adhesion to BSA±s.e.m. is also noted for each cell line. 100%=maximal absorbance at 595 nm for each cell line and each set of ECM proteins.

Figure 2

Type I collagen promotes maximal pancreatic cancer cell proliferation. The 96-well polystyrene culture dishes, not treated for tissue culture, were coated with type I collagen, type IV collagen, fibronectin, laminin, or vitronectin as described in Materials and Methods. Twenty-four hour, serum-starved MiaPaCa-2, AsPC-1, BxPC-3, CFPAC, and FG cells (5 × 10³ well⁻¹) were cultured under serum-free conditions on the indicated ECM substrates over a 4-day time course. At the indicated time points, triplicate determinations were quantified for each cell line on each substrate by measuring the absorbance at 450 nm and subtracting the value obtained at initial seeding (T=0) using CellTiter 96 Aqueous One Solution Cell Proliferation Assay™ reagent according to the manufacturer's instructions (Promega, Madison, WI, USA). Data presented represent the mean±s.e.m. from two independent experiments.

Figure 3

Type I collagen promotes maximal pancreatic cancer cell migration. Pore polycarbonate membrane filters (8 μm) were coated with either type I collagen, type IV collagen, fibronectin, laminin, or vitronectin, at 5 μg ml⁻¹. Lower chambers of the modified Boyden chamber were filled with 30 μl well⁻¹ of serum-free DMEM supplemented with 1 mg ml⁻¹ BSA, the coated filters were then placed on top of the lower chambers, and the upper chambers were secured in place. Upper chambers were filled with 5 × 10⁴ FG, AsPC-1, MiaPaCa-2, BxPC-3, or CFPAC cells that were serum-starved 24 h prior to assay, in 50 μl of the same media described above. The entire apparatus was then incubated for 24 h at 37°C. After the incubation period, the filters were fixed in methanol and stained with 0.5% toluidine blue in 3.7% formaldehyde. Excess stain was washed away with water, the attached cells on the upper side of the filters were mechanically removed using wet, cotton-tipped applicators, and the migratory cells on the underside of the filters were quantitated by counting four high-powered fields (× 100 magnification) per well using an inverted light microscope (Olympus BH 2). Results presented represent the mean±s.e.m. from two independent experiments with at least three replicates per substrate for each cell line. 100%=maximal cell number for each cell line and each set of ECM proteins.

Figure 4

Integrin, E-cadherin, and β-catenin expression in pancreatic cancer cell lines. Immunoprecipitations were conducted using 4 μg anti-integrin antibodies and 400 μg cell-surface-biotinylated extracts from pancreatic cancer cell lines as described in Materials and Methods. Immunoprecipitates were separated on 12% Nu–PAGE gels under nonreducing conditions, and protein bands were visualised using streptavidin–HRP and chemiluminescence. Autoradiograms for each particular integrin subunit or integrin heterodimer for each cell line are indicated in the right-hand margin.

Figure 5

The α₂β₁ integrin mediates pancreatic cancer cell adhesion and haptokinetic migration on type I collagen. Inhibition of AsPC-1, BxPC-3, CFPAC, and FG cell adhesion and migration assays were conducted using 96-well plates or 8 μM polycarbonate membranes coated with 5 μg ml⁻¹ type I collagen as described in Materials and Methods. Function-blocking monoclonal antibodies directed against the indicated integrins or integrin subunits were added at final concentrations ranging between 25 and 50 μg ml⁻¹ as described in Materials and Methods for each antibody and each cell line. (A) Cell adhesion and (B) haptokinetic cell migration.