Regulation of β-catenin by t-DARPP in upper gastrointestinal cancer cells.

Vangamudi B, Zhu S, Soutto M, Belkhiri A, El-Rifai W
Mol Cancer. 2011 10: 32

PMID: 21447180 · PMCID: PMC3075216 · DOI:10.1186/1476-4598-10-32

BACKGROUND - Truncated dopamine and cyclic-AMP-regulated phosphoprotein (t-DARPP) is frequently overexpressed in gastrointestinal malignancies. In this study, we examined the role of t-DARPP in regulating β-catenin.

RESULTS - The pTopFlash construct that contains multiple TCF/LEF-binding sites was used as a measure of β-catenin/TCF transcription activity. Gastric (AGS, MKN28) and esophageal (FLO-1) adenocarcinoma cancer cell lines that lack t-DARPP expression were utilized to establish stable and transient in vitro expression models of t-DARPP. The expression of t-DARPP led to a significant induction of the pTOP reporter activity, indicative of activation of β-catenin/TCF nuclear signaling. Immunofluorescence assays supported this finding and showed accumulation and nuclear translocation of β-catenin in cells expressing t-DARPP. These cells had a significant increase in their proliferative capacity and demonstrated up-regulation of two transcription targets of β-catenin/TCF: Cyclin D1 and c-MYC. Because phosphorylated GSK-3β is inactive and loses its ability to phosphorylate β-catenin and target it towards degradation by the proteasome, we next examined the levels of phospho-GSK-3β. These results demonstrated an increase in phospho-GSK-3β and phospho-AKT. The knockdown of endogenous t-DARPP in MKN45 cancer cells demonstrated a reversal of the signaling events. To examine whether t-DARPP mediated GSK-3β phosphorylation in an AKT-dependent manner, we used a pharmacologic inhibitor of PI3K/AKT, LY294002, in cancer cells expressing t-DARPP. This treatment abolished the phosphorylation of AKT and GSK-3β leading to a reduction in β-catenin, Cyclin D1, and c-MYC protein levels.

CONCLUSIONS - Our findings demonstrate, for the first time, that t-DARPP regulates β-catenin/TCF activity, thereby implicating a novel oncogenic signaling in upper gastrointestinal cancers.

MeSH Terms (27)

beta Catenin Binding Sites Blotting, Western Cell Line, Tumor Cell Nucleus Cell Proliferation Chromones Dopamine and cAMP-Regulated Phosphoprotein 32 Enzyme Inhibitors Gastrointestinal Neoplasms Gene Expression Regulation, Neoplastic Glycogen Synthase Kinase 3 Glycogen Synthase Kinase 3 beta Humans Luciferases Microscopy, Fluorescence Morpholines Mutation Phosphatidylinositol 3-Kinases Phosphoinositide-3 Kinase Inhibitors Phosphorylation Protein Isoforms Proto-Oncogene Proteins c-akt RNA Interference Signal Transduction TCF Transcription Factors Upper Gastrointestinal Tract

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