Characterization and Cloning of the Human Perlecan Promoter Region

Mary C. Farach-CarsonProstate cancer metastasizes preferentially to bone. The bone microenvironment presents the invading cells with a rich supply of growth and angiogenic factors. Because trabecular bone is in a state of resorption and deposition, the mineralized matrix is degraded and reformed constantly. This process also releases important growth factors, such as TGF-β, which may aid the survival of metastatic prostate cancer cells. Unpublished data from members of this lab group shows a large upregulation of a heparan sulfate proteoglycan, called perlecan, in the reactive stroma surrounding prostate epithelial cells. Perlecan is a structural protein located in the basement membranes and the matrix surrounding endothelial, mesenchymal and stromal cells. Among other properties, perlecan, through the heparan sulfate side chains, can bind growth factors. This property of perlecan identifies it as a protein that may help promote prostate cancer metastasis by providing the mobile cells with a scaffold to store growth and angiogenic factors in close proximity to their receptors. My project was concerned with the large upregulation of perlecan in the reactive stroma. I began my project by characterizing the promoter region for perlecan and identifying conserved transcription factor binding sites that could participate in transcriptionally regulating perlecan in prostate cancer. I identified several transcription factor binding sites of interest for further study, including NF-κB [-2410 to -2398], CREB ([-1797 to -1777] and [-709 to -689]), Smad3 ([-1301 to -1293] and [-187 to -179]), Elk-1 [-1699 to -1679], c-Jun ([-2453 to -2441] and [-2496 to -2476]) and TCF/LEF-1 ([-1521 to -1505] and [-1247 to -1231]). I then attempted to clone the promoter region from genomic DNA using polymerase chain reaction, and encountered several issues. I attempted to alter the reaction conditions and to try different kits to correct the problems. I found that addition of dimethyl sulfoxide to the reaction increased the specificity of the reaction and allowed for the successful cloning of the perlecan promoter region into a plasmid vector. Following the cloning of the vector, I began testing the effects of two growth factors, TGF-β and TNF-α, on perlecan transcription. Following treatment for 24 hours, RNA was extracted from HS27a bone marrow stromal cells and used to conduct quantitative PCR in order to test the levels of perlecan transcript. Although the data have not yet been analyzed, the cell cultures showed growth changes, namely the formation of a cobblestone growth pattern, which indicated that the growth factors affected some cellular processes. Further research needs to be conducted in order to determine if this effect indicates a change in perlecan transcription in order to determine whether perlecan could be a viable target for new therapies.This research was supported by the P01 CA098912 grant to Dr. Mary C. Farach-Carson and in part by The Howard Hughes Medical Institute.Biological Science