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

Bhavatarini Vangamudi, Shoumin Zhu, Mohammed Soutto, Abbes Belkhiri, Wael El-Rifai

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Article number32
JournalMolecular Cancer
Volume10
DOIs
StatePublished - Mar 29 2011
Externally publishedYes

Fingerprint

Catenins
Gastrointestinal Neoplasms
Phosphoproteins
Cyclic AMP
Dopamine
Glycogen Synthase Kinase 3
Cyclin D1
Neoplasms
Phosphorylation
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Proteasome Endopeptidase Complex
Phosphatidylinositol 3-Kinases
Fluorescent Antibody Technique
Stomach
Adenocarcinoma
Up-Regulation
Binding Sites
Cell Line

ASJC Scopus subject areas

  • Molecular Medicine
  • Oncology
  • Cancer Research

Cite this

Regulation of β-catenin by t-DARPP in upper gastrointestinal cancer cells. / Vangamudi, Bhavatarini; Zhu, Shoumin; Soutto, Mohammed; Belkhiri, Abbes; El-Rifai, Wael.

In: Molecular Cancer, Vol. 10, 32, 29.03.2011.

Research output: Contribution to journalArticle

Vangamudi, Bhavatarini ; Zhu, Shoumin ; Soutto, Mohammed ; Belkhiri, Abbes ; El-Rifai, Wael. / Regulation of β-catenin by t-DARPP in upper gastrointestinal cancer cells. In: Molecular Cancer. 2011 ; Vol. 10.
@article{9ee71d3c09fa4f738bdf0636ebce5389,
title = "Regulation of β-catenin by t-DARPP in upper gastrointestinal cancer cells",
abstract = "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.",
author = "Bhavatarini Vangamudi and Shoumin Zhu and Mohammed Soutto and Abbes Belkhiri and Wael El-Rifai",
year = "2011",
month = "3",
day = "29",
doi = "10.1186/1476-4598-10-32",
language = "English (US)",
volume = "10",
journal = "Molecular Cancer",
issn = "1476-4598",
publisher = "BioMed Central",

}

TY - JOUR

T1 - Regulation of β-catenin by t-DARPP in upper gastrointestinal cancer cells

AU - Vangamudi, Bhavatarini

AU - Zhu, Shoumin

AU - Soutto, Mohammed

AU - Belkhiri, Abbes

AU - El-Rifai, Wael

PY - 2011/3/29

Y1 - 2011/3/29

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=79953113067&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79953113067&partnerID=8YFLogxK

U2 - 10.1186/1476-4598-10-32

DO - 10.1186/1476-4598-10-32

M3 - Article

C2 - 21447180

AN - SCOPUS:79953113067

VL - 10

JO - Molecular Cancer

JF - Molecular Cancer

SN - 1476-4598

M1 - 32

ER -