title: Regulation of CXCR4-mediated invasion by DARPP-32 in gastric cancer cells.
abstract: Although Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) is overexpressed in two-thirds 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-overexpressing AGS cells 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 soluble MMPs, we detected a robust increase in MMP-2 activity in DARPP-32-overexpressing cells. The knockdown of endogenous DARPP-32 in the MKN-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 that DARPP-32 and CXCR4 coexist in the same protein complex. DARPP-32 prolonged the CXCR4 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.
authors.affiliation: Vanderbilt University Medical Center, 1255 Light Hall, 2215 Garland Avenue, Nashville, TN 37232, USA.
authors.forename: Shoumin
authors.initials: S
authors.lastname: Zhu
authors.forename: Jun
authors.initials: J
authors.lastname: Hong
authors.forename: Manish K
authors.initials: MK
authors.lastname: Tripathi
authors.forename: Vikas
authors.initials: V
authors.lastname: Sehdev
authors.forename: Abbes
authors.initials: A
authors.lastname: Belkhiri
authors.forename: Wael
authors.initials: W
authors.lastname: El-Rifai
mesh_terms: D045744:Cell Line, Tumor; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D053511:Matrix Metalloproteinase 14 / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D009361:Neoplasm Invasiveness; D019718:Receptors, CXCR4 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013274:Stomach Neoplasms / Q000378:metabolism* / Q000473:pathology*
chemical_list: C527276:CXCR4 protein, human; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; C497928:PPP1R1B protein, human; D019718:Receptors, CXCR4; D020778:Matrix Metalloproteinase 2; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14