Purpose: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and devastating disease. It is characterized by invasiveness, rapid progression and profound resistance to treatment, resulting in a median survival of 6 months. PDX-1, a transcription factor, is essential to the early development of the pancreas. In an adult the PDX-1 gene is turned off in the exocrine pancreas stopping further formation of the pancreas. However, PDX-1 expression is reinitiated in PDAC cells. Matrix Metalloproteinase-7 (MMP-7) is not present in normal tissues but is found in tumor cells of benign and invasive tumors of the breast, intestine, prostate, and pancreas. Because, both MMP-7 and PDX-1 are expressed in PDAC, we set out to test the hypothesis that PDX-1 positively regulates MMP-7 expression by activating the MMP-7 promoter.Methods: 100,000 SW480 tumor cells were plated in a 24 well plate. The cells were then transfected with 300ng of MMP-7 promoter/luciferase reporter, 20ng of CMVRL, and 300ng of CMV PDX-1 expression vector using Superfect reagent (Qiagen) according to the manufacture's directions. The transfection mix was left on the cells overnight. The next day the media was changed to IMEM media supplemented with 10% FBS and this was left on the cells for four hours. To measure the amount of transcription Cells were then lysed and analyzed with the Dual Assay Luciferase® assay. All transfections were performed in triplicate. Results: Using constructs containing various lengths of the MMP-7 promoter(Fig2) cloned upstream of the firefly luciferase reporter, our results show that overexpression of PDX-1 does activate the 2.3kb MMP-7 promoter. Using a map of the MMP-7 promoter gene we compared the results to determine the sequences that were necessary for activation. Our results also show that PDX-1 does indeed increase MMP-7 promoter activity by approximately 12 fold. Conclusions: These experiments show how the transcription factor PDX-1 does indeed up-regulate the MMP-7 promoter activity by approximately 12-fold. This was proven by performing a transfection and a dual luciferase reporter (promega) with various constructs of the MMP-7 promoter/reporter. The results from the reporter and the map of the MMP-7 gene support that there is a positive transcriptional element for PDX-1 found on the MMP-7 promoter. According a map of the promoter there are three possible sites for the PDX-1 protein and compared with the results of the transfection. We found three possible sites of PDX-1 binding with our MMP-7 constructs showing how PDX-1 does positively regulate the MMP-7 expression in PDAC.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]