Fibroblasts stimulate pancreatic tumor cell invasion by up regulating MMP-9 activity

Abstract

MMP-9 activity is determined by a balance between the production of MMP-9 and tissue inhibitor of metalloproteinases-2 (TIMP-2). We have previously shown that increased MMP-9 activity stimulates pancreatic cancer cell invasion and that MMP-9 localizes to stromal fibroblasts in pancreatic cancer. We now hypothesize that fibroblasts stimulate tumor cell invasion by up regulating MMP-9 activity. Methods. MMP-9 and TIMP-2 expression in PANC-1 cancer cells, fibroblasts, and PANC-1/fibroblast cocultures were determined by ELISA and Western blot. The effect of fibroblasts on PANC-1 cancer cell invasion was evaluated using a Boyden Chamber invasion assay with and without neutralizing antibodies (Ab) against MMP-9 and TIMP-2. Statistical significance was determined by ANOVA and Student’s unpaired t-test. Results. There was no significant difference in MMP-9 production among PANC-1 cancer cells, fibroblasts, and PANC-1/fibroblast cocultures ( P = 0.25). Fibroblasts produced seven times higher amounts of TIMP-2 than PANC-1 cells (28.01 ± 1.21 versus 4.03 ± 0.01 ng/ml, P < 0.02). When cocultured with PANC-1 cells, overall TIMP-2 expression was reduced to 4.65 ± 0.23 ( P < 0.02) as demonstrated by ELISA and confirmed by Western blot (see Figure 1). Fibroblasts induced a 3-fold increase in PANC-1 tumor cell invasion. Fibroblast-induced PANC-1 tumor cell invasion was inhibited by an anti-MMP-9 Ab and up regulated by an anti-TIMP-2 Ab (see Figure 2, P < 0.02 compared to fibroblasts alone). Conclusion. Fibroblasts co-cultured with pancreatic cancer cells decrease overall TIMP-2 expression. The resultant increase in MMP-9 activity promotes tumor cell invasion.