Abstract
BackgroundTruncated 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.ResultsThe 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.ConclusionsOur findings demonstrate, for the first time, that t-DARPP regulates β-catenin/TCF activity, thereby implicating a novel oncogenic signaling in upper gastrointestinal cancers.
Highlights
Truncated dopamine and cyclic-AMP-regulated phosphoprotein (t-DARPP) is frequently overexpressed in gastrointestinal malignancies
Molecular investigation of critical target genes at 17q12 amplicon in gastric adenocarcinoma has led to the identification of DARPP-32 and t-DARPP, a truncated isoform of DARPP-32, as two novel cancer-related genes [13]. t-DARPP is frequently overexpressed in several human adenocarcinomas such as those of the stomach, colon, esophagus, breast, and prostate [14,15,16,17,18]
We have reported that t-DARPP can regulate β-catenin/TCF signaling in upper gastrointestinal cancer cells
Summary
Truncated dopamine and cyclic-AMP-regulated phosphoprotein (t-DARPP) is frequently overexpressed in gastrointestinal malignancies. Upper gastrointestinal adenocarcinomas (UGCs) are among the most prevalent causes of cancer-related deaths in the world. This category of cancers includes adenocarcinomas of the stomach, gastroesophageal junction (GEJ), and lower esophagus. Chemotherapy is currently one of the primary options for treatment of gastric cancer, it often provides poor clinical prognosis due to the underlying resistance mechanisms [10,11]. Limited understanding of such inherent protective mechanisms enforces a need to identify novel signaling pathways that can possibly reveal novel drug targets towards the development of advanced therapeutic alternatives. The molecular signaling mechanisms governing tDARPP’s biological functions remain fairly unexplored
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