Regulation of CXCR4-mediated invasion by DARPP-32 in gastric cancer cells

Shoumin Zhu, Jun Hong, Manish K. Tripathi, Vikas Sehdev, Abbes Belkhiri, Wael El-Rifai

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Although Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) is overexpressed in twothirds of gastric cancers, its impact on molecular functions has not been fully characterized. In this study, we examined the role of DARPP-32 in gastric cancer cell invasion. Using matrigel-coated Boyden chamber invasion assay,DARPP-32-overexpressingAGScells showed a three-fold increase in invasion relative to the vector control (P < 0.01). We also tested the transendothelial cell invasion as a measure of cell aggressiveness using the impedance-based human umbilical vein endothelial cells invasion assay and obtained similar results (P < 0.001). Western blot analysis indicated that overexpression of DARPP-32 mediated an increase in the membrane-type 1 matrix metalloproteinase (MT1-MMP) and CXCR4 protein levels. Consistent with the role of MT1-MMP in cleaving extracellular matrix proteins initiating the activation of solubleMMPs, we detected a robust increase inMMP-2 activity in DARPP-32- overexpressing cells. The knockdown of endogenousDARPP-32 in theMKN-45 cells reversed these signaling events and decreased cell invasive activity. We tested whether the invasive activity mediated by DARPP-32 might involve sustained signaling via CXCR4-dependent activation of the MT1-MMP/MMP-2 pathway. The small-molecule CXCR4 antagonist (AMD3100) and CXCR4-siRNA blocked DARPP-32-induced cell invasion. We further examined our hypothesis that DARPP-32 could interact with CXCR4 and stabilize its levels following stimulation with its ligand, CXCL12. Using reciprocal coimmunoprecipitation and immunofluorescence experiments, we found thatDARPP-32 andCXCR4 coexist in the same protein complex.DARPP-32 prolonged theCXCR4 protein half-life and reduced ubiquitination of the CXCR4 protein, following treatment with its ligand, CXCL12. In conclusion, these findings show a novel mechanism by which DARPP-32 promotes cell invasion by regulating CXCR4-mediated activation of the MT1-MMP/MMP-2 pathway.

Original languageEnglish (US)
Pages (from-to)86-94
Number of pages9
JournalMolecular Cancer Research
Volume11
Issue number1
DOIs
StatePublished - Jan 1 2013
Externally publishedYes

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Dopamine and cAMP-Regulated Phosphoprotein 32
Stomach Neoplasms
Matrix Metalloproteinase 14
Matrix Metalloproteinase 2
Matrix Metalloproteinases
Proteins
Ligands
Extracellular Matrix Proteins
Ubiquitination
Human Umbilical Vein Endothelial Cells
Electric Impedance
Small Interfering RNA
Fluorescent Antibody Technique
Half-Life

ASJC Scopus subject areas

  • Molecular Biology
  • Oncology
  • Cancer Research

Cite this

Regulation of CXCR4-mediated invasion by DARPP-32 in gastric cancer cells. / Zhu, Shoumin; Hong, Jun; Tripathi, Manish K.; Sehdev, Vikas; Belkhiri, Abbes; El-Rifai, Wael.

In: Molecular Cancer Research, Vol. 11, No. 1, 01.01.2013, p. 86-94.

Research output: Contribution to journalArticle

Zhu, Shoumin ; Hong, Jun ; Tripathi, Manish K. ; Sehdev, Vikas ; Belkhiri, Abbes ; El-Rifai, Wael. / Regulation of CXCR4-mediated invasion by DARPP-32 in gastric cancer cells. In: Molecular Cancer Research. 2013 ; Vol. 11, No. 1. pp. 86-94.
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abstract = "Although Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) is overexpressed in twothirds of gastric cancers, its impact on molecular functions has not been fully characterized. In this study, we examined the role of DARPP-32 in gastric cancer cell invasion. Using matrigel-coated Boyden chamber invasion assay,DARPP-32-overexpressingAGScells showed a three-fold increase in invasion relative to the vector control (P < 0.01). We also tested the transendothelial cell invasion as a measure of cell aggressiveness using the impedance-based human umbilical vein endothelial cells invasion assay and obtained similar results (P < 0.001). Western blot analysis indicated that overexpression of DARPP-32 mediated an increase in the membrane-type 1 matrix metalloproteinase (MT1-MMP) and CXCR4 protein levels. Consistent with the role of MT1-MMP in cleaving extracellular matrix proteins initiating the activation of solubleMMPs, we detected a robust increase inMMP-2 activity in DARPP-32- overexpressing cells. The knockdown of endogenousDARPP-32 in theMKN-45 cells reversed these signaling events and decreased cell invasive activity. We tested whether the invasive activity mediated by DARPP-32 might involve sustained signaling via CXCR4-dependent activation of the MT1-MMP/MMP-2 pathway. The small-molecule CXCR4 antagonist (AMD3100) and CXCR4-siRNA blocked DARPP-32-induced cell invasion. We further examined our hypothesis that DARPP-32 could interact with CXCR4 and stabilize its levels following stimulation with its ligand, CXCL12. Using reciprocal coimmunoprecipitation and immunofluorescence experiments, we found thatDARPP-32 andCXCR4 coexist in the same protein complex.DARPP-32 prolonged theCXCR4 protein half-life and reduced ubiquitination of the CXCR4 protein, following treatment with its ligand, CXCL12. In conclusion, these findings show a novel mechanism by which DARPP-32 promotes cell invasion by regulating CXCR4-mediated activation of the MT1-MMP/MMP-2 pathway.",
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AU - Hong, Jun

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AU - El-Rifai, Wael

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N2 - Although Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) is overexpressed in twothirds of gastric cancers, its impact on molecular functions has not been fully characterized. In this study, we examined the role of DARPP-32 in gastric cancer cell invasion. Using matrigel-coated Boyden chamber invasion assay,DARPP-32-overexpressingAGScells showed a three-fold increase in invasion relative to the vector control (P < 0.01). We also tested the transendothelial cell invasion as a measure of cell aggressiveness using the impedance-based human umbilical vein endothelial cells invasion assay and obtained similar results (P < 0.001). Western blot analysis indicated that overexpression of DARPP-32 mediated an increase in the membrane-type 1 matrix metalloproteinase (MT1-MMP) and CXCR4 protein levels. Consistent with the role of MT1-MMP in cleaving extracellular matrix proteins initiating the activation of solubleMMPs, we detected a robust increase inMMP-2 activity in DARPP-32- overexpressing cells. The knockdown of endogenousDARPP-32 in theMKN-45 cells reversed these signaling events and decreased cell invasive activity. We tested whether the invasive activity mediated by DARPP-32 might involve sustained signaling via CXCR4-dependent activation of the MT1-MMP/MMP-2 pathway. The small-molecule CXCR4 antagonist (AMD3100) and CXCR4-siRNA blocked DARPP-32-induced cell invasion. We further examined our hypothesis that DARPP-32 could interact with CXCR4 and stabilize its levels following stimulation with its ligand, CXCL12. Using reciprocal coimmunoprecipitation and immunofluorescence experiments, we found thatDARPP-32 andCXCR4 coexist in the same protein complex.DARPP-32 prolonged theCXCR4 protein half-life and reduced ubiquitination of the CXCR4 protein, following treatment with its ligand, CXCL12. In conclusion, these findings show a novel mechanism by which DARPP-32 promotes cell invasion by regulating CXCR4-mediated activation of the MT1-MMP/MMP-2 pathway.

AB - Although Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) is overexpressed in twothirds of gastric cancers, its impact on molecular functions has not been fully characterized. In this study, we examined the role of DARPP-32 in gastric cancer cell invasion. Using matrigel-coated Boyden chamber invasion assay,DARPP-32-overexpressingAGScells showed a three-fold increase in invasion relative to the vector control (P < 0.01). We also tested the transendothelial cell invasion as a measure of cell aggressiveness using the impedance-based human umbilical vein endothelial cells invasion assay and obtained similar results (P < 0.001). Western blot analysis indicated that overexpression of DARPP-32 mediated an increase in the membrane-type 1 matrix metalloproteinase (MT1-MMP) and CXCR4 protein levels. Consistent with the role of MT1-MMP in cleaving extracellular matrix proteins initiating the activation of solubleMMPs, we detected a robust increase inMMP-2 activity in DARPP-32- overexpressing cells. The knockdown of endogenousDARPP-32 in theMKN-45 cells reversed these signaling events and decreased cell invasive activity. We tested whether the invasive activity mediated by DARPP-32 might involve sustained signaling via CXCR4-dependent activation of the MT1-MMP/MMP-2 pathway. The small-molecule CXCR4 antagonist (AMD3100) and CXCR4-siRNA blocked DARPP-32-induced cell invasion. We further examined our hypothesis that DARPP-32 could interact with CXCR4 and stabilize its levels following stimulation with its ligand, CXCL12. Using reciprocal coimmunoprecipitation and immunofluorescence experiments, we found thatDARPP-32 andCXCR4 coexist in the same protein complex.DARPP-32 prolonged theCXCR4 protein half-life and reduced ubiquitination of the CXCR4 protein, following treatment with its ligand, CXCL12. In conclusion, these findings show a novel mechanism by which DARPP-32 promotes cell invasion by regulating CXCR4-mediated activation of the MT1-MMP/MMP-2 pathway.

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