Impact of the secretome of activated pancreatic stellate cells on growth and differentiation of pancreatic tumour cells

Abstract

Pancreatic ductal adenocarcinoma (PDAC) exists in a complex desmoplastic microenvironment. As part of it, pancreatic stellate cells (PSCs) provide a fibrotic niche, stimulated by a dynamic communication between activated PSCs and tumour cells. Investigating how PSCs contribute to tumour development and for identifying proteins that the cells secrete during cancer progression, we studied by means of complex antibody microarrays the secretome of activated PSCs. A large number of secretome proteins were associated with cancer-related functions, such as cell apoptosis, cellular growth, proliferation and metastasis. Their effect on tumour cells could be confirmed by growing tumour cells in medium conditioned with activated PSC secretome. Analyses of the tumour cells' proteome and mRNA revealed a strong inhibition of tumour cell apoptosis, but promotion of proliferation and migration. Many cellular proteins that exhibited variations were found to be under the regulatory control of eukaryotic translation initiation factor 4E (eIF4E), whose expression was triggered in tumour cells grown in the secretome of activated PSCs. Inhibition by an eIF4E siRNA blocked the effect, inhibiting tumour cell growth in vitro. Our findings show that activated PSCs acquire a pro-inflammatory phenotype and secret proteins that stimulate pancreatic cancer growth in an eIF4E-dependent manner, providing further insight into the role of stromal cells in pancreatic carcinogenesis and cancer progression.

Figure 1

Scheme of the overall experimental set-up. First, the protein content of the secretome of activated PSCs was analysed. On this basis, predictions were made about the functional consequences, which the secreted proteins would have in recipient cells. Second, PT45P1 tumour cells were grown in media conditioned with secretome. The intracellular proteome was studied and again used for functional predictions. Both the predictions from secretome and intracellular proteome were compared and validated by investigating the actual functional variations observed and by identifying relevant regulative factors.

Figure 2

Verification of PSC secreted proteins by immunoblot and ELISA. (A) Western blotting of conditioned media concentrates from activated or quiescent PSCs; enolase (ENO-1) was used as control protein. (B) Bar chart of the relative abundance in secretome of non-activated or activated PSCs, respectively, of fibronectin 1 (FN-1) and collagen-1 (Col-1); detection was by commercial ELISA.

Figure 3

The impact of PSC secretome on PDAC cells. Serum-starved PT45P1 cells were treated with secretome from non-activated or activated PSC; the abundance of key molecules of cell apoptosis and viability were measured in the PT45P1 cellular proteome by using Western blots (A). Variations in apoptosis were studied by two different assays (B) measuring the mitochondrial membrane potential (top) or the caspase-3/7 activity (bottom). In (C), results from Western blots are shown that look at proteins relevant for differentiation.

Figure 4

Proliferation and migration assays with PT45P1 cells upon growth in activated or non-activated PSC secretome. In (A), the variation of proliferation after 48 h is shown. Below (B), the effect on cell migration after 24 h is presented using a scratch assay.

Figure 5

Schematic representation of the regulative function of eIF4E. Using the IPA analysis software, effects are shown of proteins of the secretome of activated PSCs on eIF4E (upstream factors) as well as the effects of eIF4E on intracellular proteins of the tumour cells. Red indicates an increase, green a decrease in abundance compared to control conditions. Orange stands for activation, blue for inhibition.

Figure 6

The effect of knocking down eIF4E protein expression by small interfering RNA. Tumour cells PT45P1 and Panc-1 were transfected with 100 nM of an eIF4E-specific siRNA or a control siRNA of scrambled sequence for 48 h, followed by serum starvation overnight. Subsequently, they were grown in presence or absence of activated PSC secretome for 24 h. The effect of the various conditions on eIF4E is shown, determined by Western blotting. Also, the results of caspase-3/7 assays are shown for each column (c1 to c6). Note: the two lanes c1 and c2 are exchanged in the Western blots generated from Panc-1 cells.

Figure 7

Transient silencing eIF4E in Capan-1 pancreatic cancer cells inhibits cell migration. Capan-1 cells were transfected with 100 nM of either eIF4E-specific siRNA or a control siRNA of scrambled sequence for 48 h. Cells were serum starved overnight and either left untreated or treated with activated PSC secretome. A gap was generated by physically scraping off cells. The gap was inspected at different time intervals of up to 48 h at the growth conditions indicated in the figure.