Effect of altering fibroblast integrin associated protein expression on the growth and protein expression of pancreas cancer cells.

Abstract

251 Background: The lack of success of current treatments for pancreatic ductal adenocarcinoma (PDA) may be due in part to the presence of the dense surrounding stromal response and the interactions between the stroma and cancer cells. We have shown that integrin associated proteins, in particular PINCH, are expressed to a higher degree in the stroma adjacent to the tumor cells and PINCH expression is positively correlated with poorer PDA patient outcomes. We hypothesize that decreasing PINCH protein expression in the tumor associated stroma will decrease the growth and expression of growth promoting proteins in PDA cells. Methods: Red fluorescent protein (RFP) stably expressing MiaPaCa-2 cells were co-cultured with WI38 fibroblasts, or WI38 fibroblasts with shRNA knockdown of PINCH protein (PINCH KD). Cell growth of the PDA cells alone and upon exposure to WI38 and PINCH KD fibroblasts was determined using RFP at 48 hours. PINCH, Akt, and pAKt protein expression in cultured PDA cells, exposed to media, WI38, or WI38 PINCH KD cells via a transwell system, were determined by western blot with values normalized to GAPDH protein expression at 48 hours. Results: MiaPaCa-2 cells grown in co-culture with WI38 cells had a 70%±5% increase in RFP, while those grown in co-culture with WI38 PINCH KD cells had a 40%±3% increase relative to MiaPaCa2 cells grown alone. In terms of relative PINCH protein expression there was an increase in MiaPaCa-2 (20%±5%) cells grown in co-culture with WI38 cells compared to alone but not when grown with PINCH KD (6%±3%). MiaPaCa-2 cells had a greater increase in AKT (47%±6% compared with 19%±4%) and pAKT (36%±5 and 22%±4%) in the cells grown with WI38 cells compared to PDA cells grown with WI38 PINCH KD. Conclusions: Reductions in fibroblast PINCH protein expression are associated with reductions in the growth of adjacent PDA cells, as well as the expression of growth enhancing proteins (PINCH, AKT, and pAKT). Since greater PINCH expression within PDA stromal cells is associated with poorer patient outcomes, understanding the mechanisms associated with this tumor-stromal interaction may provide intervention opportunities.