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

Taylor A. Sohn, Gloria H. Su, Byungwoo Ryu, Charles J. Yeo, Scott E. Kern

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Objective To screen a library of small chemicals for compounds that activate the DPC4 signal transduction pathway in a human pancreatic cancer cell line. Summary Background Data Various tumor-suppressor genes are mutated in all human 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-β (TGFβ) signaling pathway. It would 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 reporter gene already incorporated, a DPC4-specific reporter construct was genetically engineered. This was done by inserting six copies of the palindromic 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 line (PANC-1) that has wild-type DPC4. Several stably transfected clones were tested for basal luciferase expression and inducibility with TGFβ, which is known to activate the DPC4 signal transduction pathway. A single transfected clone was chosen for the drug screen based on basal luciferase (reporter) expression and TGFβ inducibility, A systematic screen of the chemical 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 system was used to screen a library of 16,320 random compounds to identify agents that conferred robust augmentation of the DPC4 signal transduction pathway. 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. The latter compound was shown to be a novel histone deacetylase inhibitor and was further characterized. Conclusions These results confirm the feasibility of a specific high-throughput reporter system to screen a large compound library in human cells efficiently. The screening identified several compounds capable of augmenting DPC4-specific luciferase reporter activity, and a specific mechanism for one compound was identified. The discovery of such agents will aid our understanding of complex tumor-suppressive signaling pathways and may identify other potential therapeutic targets within this critical signaling pathway. In addition, random drug screening provides an unbiased method for identifying drugs or lead compounds for potential therapeutic use.

Original languageEnglish (US)
Pages (from-to)696-703
Number of pages8
JournalAnnals of Surgery
Volume233
Issue number5
DOIs
StatePublished - 2001
Externally publishedYes

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Preclinical Drug Evaluations
Luciferases
Pancreatic Neoplasms
Signal Transduction
Transforming Growth Factors
Small Molecule Libraries
Neoplasms
Tumor Suppressor Genes
Plasmids
Clone Cells
Cell Line
Histone Deacetylase Inhibitors
Critical Pathways
Therapeutic Uses
Human Genome
Therapeutics
Reporter Genes
Pharmaceutical Preparations
Libraries
Adenocarcinoma

ASJC Scopus subject areas

  • Surgery

Cite this

High-throughput drug screening of the DPC4 tumor-suppressor pathway in human pancreatic cancer cells. / Sohn, Taylor A.; Su, Gloria H.; Ryu, Byungwoo; Yeo, Charles J.; Kern, Scott E.

In: Annals of Surgery, Vol. 233, No. 5, 2001, p. 696-703.

Research output: Contribution to journalArticle

Sohn, Taylor A. ; Su, Gloria H. ; Ryu, Byungwoo ; Yeo, Charles J. ; Kern, Scott E. / High-throughput drug screening of the DPC4 tumor-suppressor pathway in human pancreatic cancer cells. In: Annals of Surgery. 2001 ; Vol. 233, No. 5. pp. 696-703.
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AU - Kern, Scott E.

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N2 - Objective To screen a library of small chemicals for compounds that activate the DPC4 signal transduction pathway in a human pancreatic cancer cell line. Summary Background Data Various tumor-suppressor genes are mutated in all human 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-β (TGFβ) signaling pathway. It would 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 reporter gene already incorporated, a DPC4-specific reporter construct was genetically engineered. This was done by inserting six copies of the palindromic 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 line (PANC-1) that has wild-type DPC4. Several stably transfected clones were tested for basal luciferase expression and inducibility with TGFβ, which is known to activate the DPC4 signal transduction pathway. A single transfected clone was chosen for the drug screen based on basal luciferase (reporter) expression and TGFβ inducibility, A systematic screen of the chemical 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 system was used to screen a library of 16,320 random compounds to identify agents that conferred robust augmentation of the DPC4 signal transduction pathway. 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. The latter compound was shown to be a novel histone deacetylase inhibitor and was further characterized. Conclusions These results confirm the feasibility of a specific high-throughput reporter system to screen a large compound library in human cells efficiently. The screening identified several compounds capable of augmenting DPC4-specific luciferase reporter activity, and a specific mechanism for one compound was identified. The discovery of such agents will aid our understanding of complex tumor-suppressive signaling pathways and may identify other potential therapeutic targets within this critical signaling pathway. In addition, random drug screening provides an unbiased method for identifying drugs or lead compounds for potential therapeutic use.

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