SILENCING OF BASIGIN BY RNA INTERFERENCE REDUCES THE TUMORIGENITY OF PANCREATIC CANCER CELLS

Abstract

Basigin (Bsg) is released by tumor cells to stimulate MMP synthesis in adjacent mesenchymal cells and has gained growing interest due to its role in cancer cell - host interaction. Because the role of Bsg in cancer cell biology is unknown, we developed an inducible siRNA expression system that is based on the tetracycline repressor and eukaryotic RNA polymerase III promoter H1 in MiaPaCa2 pancreatic cancer cells to investigate the Bsg loss of function phenotye in MiaPaCa2 with respect to invasion and tumorigenity. Wild type MiaPaCa2 strongly express and release Basigin as assessed by rtPCR, western-blotting, immunoassay and fluorescence-microscopy. RNAi reduced the expression of Basigin mRNA as well as cell associated and soluble Bsg protein by more than 90%. In the presence of 10% fetal calf serum Basigin silencing reduced adhesion dependent tumor cell proliferation in culture whereas only a modest reduction of colony formation in soft-agar growth was observed. The reduced growth rate correlated with a reduction of the BrdU labeling index (−30%). Moreover, Basigin knock-out reduced invasiveness of cancer-cells in a boyden chamber assay and in the CAM-Assay. Finally, Basigin silencing reduced the size of subcutaneously induced cancer-cell xenografts in nude-mice grown for 6 weeks by almost 50%. To elucidate the genes involved in the Bsg knock-out phenotype, we employed a whole genome DNA microarry. Using stringent selection criteria we identified 913 genes that were differentially expressed after tetracylcine induced bsg silencing compared to the uninduced counterpart. The differentially regulated genes were significantly overrepresented in Gene Ontology groups linked to regulation of signal transduction, cell-growth and apoptosis. In conclusion we identified the Basigin protein as a major regulator of cell growth and invasion in pancreatic cancer. Moreover, our results imply the therapeutic potential of RNAi in the treatment of pancreatic cancer by targeting overexpressed genes like Basigin.