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

Various tumor-suppressor genes are mutated in all human cancers. DPC4 (deleted in pancreatic carcinoma, locus 4), also known as MADH4/SMAD4, is a tumor-suppressor gene located on chromosome 18q21.1. It is known to be inactivated in approximately 50% of human pancreatic adenocarcinomas, 1,2 as well as in many biliary 3,4 and colorectal cancers. 5,6 The DPC4 protein belongs to the evolutionarily conserved family of Smad proteins that serve as crucial intracellular mediators of the transforming growth factor-β (TGFβ) signaling pathway. The pathway is summarized in Figure 1. All members of the TGFβ superfamily of cytokines signal through membrane-bound serine threonine kinase receptor complexes. TGFβ and related ligands bind to their cell membrane receptors, which in turn phosphorylate the receptor-regulated Smad proteins, Smads 1 to 3. These phosphorylated Smads then form heteromeric complexes with DPC4 (specifically Smad2 and Smad3). These heteromeric complexes are translocated to the nucleus, where DPC4 binds a specific DNA sequence, the Smad-binding element (SBE), functioning as a transcriptional regulator. 7–10 Figure 1. The transforming growth factor-β (TGFβ)/DPC4 pathway. TGFβ binds to its cell surface receptor, a serine threonine kinase, which then phosphorylates Smads 1 to 3. Once phosphorylated, Smad2 and Smad3 form heteromeric complexes ... TGFβ can play complex roles in tumorigenesis, suppressing carcinogenesis in the early stages and acting as a tumor promoter in later stages. TGFβ is a potent inhibitor of epithelial cell growth, whose downstream functions include induction of apoptosis and cell-cycle arrest. Loss of DPC4 during tumor progression relieves this inhibition through a variety of mechanisms. 11 Reexpression of the DPC4 protein in vitro 12–16 and in vivo 11 has been shown to restore TGFβ signaling, induce apoptosis, and lead to growth inhibition. Therefore, it would be useful to identify therapies that augment or restore the downstream functions of this critical signal transduction pathway. The current methods commonly used to elucidate signal transduction pathways involve physical interactions (two-hybrid analysis or protein affinity chromatography), experimental genetic means (yeast or bacterial mutagenesis), and observations of natural genetic variance (human tumors or inherited disease susceptibility). Each of these methods has its limitations and strengths. Screening of random chemical libraries can encompass all the benefits of current methodologies. Such screening is both unbiased and high-throughput, meaning it can be performed efficiently on large numbers of compounds. Moreover, it can be readily applied to probe complex biologic systems in mammalian cells, making it applicable to human disease. 17 In addition, random screening of chemical libraries may identify drugs or lead compounds for potential therapeutic use in human cancers. This report confirms the feasibility of a specific high-throughput reporter system to screen a large compound library in human pancreatic cancer cells. The screening identified several chemicals capable of augmenting and inhibiting DPC4-specific luciferase reporter activity.