Correlation with different motility and metastatic potential in pancreatic cancer cell lines

Background: the aims of this study were to gain insights on the molecular features of pancreatic cancer metastatic phenotype using a cell model derived from SUIT-2 pancreatic adenocarcinoma cell line: S2m (motile, non metastatic) and S2CP9 (motile, highly metastatic). We focused on: 1) the signaling that regulate cell motility and migration using functional interference approaches based on cell permeable inhibitory peptides of RhoA and ζPKC enzymes; 2) the identification of the proteins released by the individual subclones. Methods: we used RhoA and ζPKC inhibitory peptides in in vitro experiments (adhesion plate, wound-healing assay and transwell assay) to evaluate the role of these enzymes. We analyzed with Multidimensional Protein Identification Technology (MudPIT) small amounts of serum-free supernatant produced by cultured SUIT-2 cells. The approach involves the generation of peptides from enzymatic digestion of a complex protein mixture, their separation by means of two micro-HPLC columns (cation exchange and reversed phase, 2DC) and direct analysis of eluted peaks by tandem mass spectrometry. The identification of the corresponding proteins is then obtained through an automated database search with appropriate software, such as the SEQUEST algorithm for data handling of mass spectra. Results: in both cell lines the combination of RhoA and ζPKC inhibitory peptides inhibited cell adhesion, although this effect was more evident in S2-CP9 cell line. In wound healing assay, we noticed that S2m was unable to migrate toward both ends of the wound even if its motility score was overlapping with that of S2-CP9. We then studied how both clones behaved in an experimental setting used to study chemotaxis, where migration of the cells occurs through a 8μm pores membrane. In this case the spontaneus migration occurs, although at different extent, in both clones and was almost completely abolished by the individual treatments with RhoA and ζPKC inhibitory peptides. Several proteins were found to be differentially released in these clones: Perlecan, Versican isoforms V0 and V3, Vimentin, Thrombospondin 1, Matrix metalloproteinases 1 and 7, HSD-2, Mac25, and uPA are among these. The most remarkable difference between non metastatic and metastatic subclones was an increased secretion of Matrix metalloproteinases and uPA, together with a decrease of proteoglycans (Perlecan, Versican V0), Thrombospondin 1 and Vimentin. Conclusions: the invasive and metastatic capability of malignancies is associated with the acquisition of anomalous motile behavior by cancer cells. Our experiments demonstrate that RhoA and ζPKC enzymes are involved in different aspects related to cell adhesion, motility on a surface or through 8 μm pores in non metastatic or metastatic S2m and S2-CP9 clones. Altogether these results indicate that motility and metastatic attitude activate overlapping, yet distinct pathways and that metastatic clones appear to be more sensitive to the disruption of cytoskeletron dynamics than highly motile but non metastatic clones. The pattern of released proteins differentiate motility and metastatic attitude, as metastatic cells release a different array of polypeptides in comparison to motile clones. The role of these products in determining, detecting or influencing metastatic behaviour need further investigations.