High-throughput drug screening of the DPC4 tumor-suppressor pathway in human pancreatic cancer cells

Objective To screen a library of small chemicals for compounds that activat e the DPC4 signal transduction pathway in a human pancreatic cancer cell li ne. Summary Background Data Various tumor-suppressor genes are mutated in all h uman cancers. Specifically, DPC4 (deleted in pancreatic carcinoma, locus 4 or MADH4/SMAD4) is a tumor-suppressor gene mutated in approximately 50% of human pancreatic adenocarcinomas. DPC4 plays an important role in the well- studied transforming growth factor-beta (TGF beta) signaling pathway. It wo uld be useful to identify therapies that augment or restore the downstream functions of this critical signal transduction pathway, in hopes that such therapy would have a rational role in anticancer therapy. Methods Using a commercially available plasmid vector with a luciferase rep orter gene already incorporated, a DPC4-specific reporter construct was gen etically engineered. This was done by inserting six copies of the palindrom ic Smad binding element (6SBE), which is a DNA binding element specific for DPC4, in front of the minimal promoter in the plasmid. This construct was then stably integrated into the genome of a human pancreatic cancer cell li ne (PANC-1) that has wild-type DPC4. Several stably transfected clones were tested for basal luciferase expression and inducibility with TGF beta, whi ch is known to activate the DPC4 signal transduction pathway. A single tran sfected clone was chosen for the drug screen based on basal luciferase (rep orter) expression and TGF beta inducibility. A systematic screen of the che mical library was then performed, using luciferase activity to detect DPC4 activity and induction of the signaling pathway. Results A high-throughput system based on this stably integrated reporter s ystem was used to screen a library of 16,320 random compounds to identify a gents that conferred robust augmentation of the DPC4 signal transduction pa thway. Of the 16,320 compounds screened, 11 were associated with a 2- to 5- fold induction of luciferase activity, and one with a 12-fold activation. T he latter compound was shown to be a novel histone deacetylase inhibitor an d was further characterized. Conclusions These results confirm the feasibility of a specific high-throug hput reporter system to screen a large compound library in human cells effi ciently. The screening identified several compounds capable of augmenting D PC4-specific luciferase reporter activity, and a specific mechanism for one compound was identified. The discovery of such agents will aid our underst anding of complex tumor-suppressive signaling pathways and may identify oth er potential therapeutic targets within this critical signaling pathway. In addition, random drug screening provides an unbiased method for identifyin g drugs or lead compounds for potential therapeutic use.