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GATA6 activates Wnt signaling in pancreatic cancer by negatively regulating the Wnt antagonist Dickkopf-1.

Zhong Y, Wang Z, Fu B, Pan F, Yachida S, Dhara M, Albesiano E, Li L, Naito Y, Vilardell F, Cummings C, Martinelli P, Li A, Yonescu R, Ma Q, Griffin CA, Real FX, Iacobuzio-Donahue CA - PLoS ONE (2011)

Bottom Line: The effects of GATA6 were found to be due to its ability to bind DNA, as forced overexpression of a DNA-binding mutant of GATA6 had no effects on cell growth in vitro or in vivo, nor did they affect Wnt signaling levels in these same cells.Transient transfection of GATA6, but not mutant GATA6, into cancer cell lines led to decreased DKK1 mRNA expression and secretion of DKK1 protein into culture media.Forced overexpression of DKK1 antagonized the effects of GATA6 on Wnt signaling in pancreatic cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins Medical Institutions, Baltimore, Maryland, United States of America.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease characterized by late diagnosis and treatment resistance. Recurrent genetic alterations in defined genes in association with perturbations of developmental cell signaling pathways have been associated with PDAC development and progression. Here, we show that GATA6 contributes to pancreatic carcinogenesis during the temporal progression of pancreatic intraepithelial neoplasia by virtue of Wnt pathway activation. GATA6 is recurrently amplified by both quantitative-PCR and fluorescent in-situ hybridization in human pancreatic intraepithelial neoplasia and in PDAC tissues, and GATA6 copy number is significantly correlated with overall patient survival. Forced overexpression of GATA6 in cancer cell lines enhanced cell proliferation and colony formation in soft agar in vitro and growth in vivo, as well as increased Wnt signaling. By contrast siRNA mediated knockdown of GATA6 led to corresponding decreases in these same parameters. The effects of GATA6 were found to be due to its ability to bind DNA, as forced overexpression of a DNA-binding mutant of GATA6 had no effects on cell growth in vitro or in vivo, nor did they affect Wnt signaling levels in these same cells. A microarray analysis revealed the Wnt antagonist Dickopf-1 (DKK1) as a dysregulated gene in association with GATA6 knockdown, and direct binding of GATA6 to the DKK1 promoter was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assays. Transient transfection of GATA6, but not mutant GATA6, into cancer cell lines led to decreased DKK1 mRNA expression and secretion of DKK1 protein into culture media. Forced overexpression of DKK1 antagonized the effects of GATA6 on Wnt signaling in pancreatic cancer cells. These findings illustrate that one mechanism by which GATA6 promotes pancreatic carcinogenesis is by virtue of its activation of canonical Wnt signaling via regulation of DKK1.

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GATA6 Overexpression Correlates with Canonical Wnt Signaling.(A) Wnt signaling activity in AsPC1-GATA6sh and A13A-GATA6sh cells based on TOPFLASH assay. Luciferase activity is represented as the ratio of OT to OF levels in cells with GATA6 knockdown relative to that of mock-transfected cells. (B) Wnt signaling activity in Panc1-GATA6 and HPNE-GATA6 cells determined by TOPFLASH assay. Luciferase activity is represented as the ratio of OT to OF levels in GATA6 transfected cells relative to that of mock-transfected cells. (C and D) Panc1-GATA6 cells were transiently transfected with ß-catenin or mock shRNA and (C) cell proliferation or (D) colony formation determined. All experimental data shown represents the summary three independent experiments. *, p<0.05; **, p<0.01. (E) Immunolabeling patterns of GATA6 and ß-catenin protein in two representative PDAC tissues. Arrows indicate nuclear labeling of both GATA6 and ß-catenin in serial sections of the same cancer tissue. By contrast, the PDAC sample with low GATA6 expression also shows no expression of ß-catenin.
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pone-0022129-g004: GATA6 Overexpression Correlates with Canonical Wnt Signaling.(A) Wnt signaling activity in AsPC1-GATA6sh and A13A-GATA6sh cells based on TOPFLASH assay. Luciferase activity is represented as the ratio of OT to OF levels in cells with GATA6 knockdown relative to that of mock-transfected cells. (B) Wnt signaling activity in Panc1-GATA6 and HPNE-GATA6 cells determined by TOPFLASH assay. Luciferase activity is represented as the ratio of OT to OF levels in GATA6 transfected cells relative to that of mock-transfected cells. (C and D) Panc1-GATA6 cells were transiently transfected with ß-catenin or mock shRNA and (C) cell proliferation or (D) colony formation determined. All experimental data shown represents the summary three independent experiments. *, p<0.05; **, p<0.01. (E) Immunolabeling patterns of GATA6 and ß-catenin protein in two representative PDAC tissues. Arrows indicate nuclear labeling of both GATA6 and ß-catenin in serial sections of the same cancer tissue. By contrast, the PDAC sample with low GATA6 expression also shows no expression of ß-catenin.

Mentions: GATA proteins are linked to Wnt signaling in embryogenesis of the heart and lungs [4], [30], [31]. We therefore hypothesized that GATA6 contributes to pancreatic carcinogenesis in part through its effects on Wnt signaling, a putative relationship that has not been explored in any detail for this tumor type. Compared to mock shRNA lentiviral-infected cells, both AsPC1-GATA6sh and A13A-GATA6sh cells showed a significant decrease in functional Wnt signaling activity by TOPFLASH assay (Figure 4A) whereas overexpression of GATA6 in Panc1 and HPNE cells promoted Wnt signaling activity (Figure 4B). To determine if ß-catenin expression is required for these effects we silenced ß-catenin expression using an shRNA strategy in Panc1-GATA6 cells, leading to a significant inhibition of cell proliferation and colony formation (Figure 4C and 4D). In pancreatic cancer tissues, GATA6 overexpression was significantly correlated with nuclear accumulation of ß-catenin protein (8/12 PDACs with GATA6 overexpression showing ß-catenin nuclear accumulation versus 3/20 without GATA6 overexpression, p = 0.004) (Figure 4E). Thus, GATA6 overexpression in PDAC contributes to cell proliferation and colony formation by enhancing canonical Wnt signaling.


GATA6 activates Wnt signaling in pancreatic cancer by negatively regulating the Wnt antagonist Dickkopf-1.

Zhong Y, Wang Z, Fu B, Pan F, Yachida S, Dhara M, Albesiano E, Li L, Naito Y, Vilardell F, Cummings C, Martinelli P, Li A, Yonescu R, Ma Q, Griffin CA, Real FX, Iacobuzio-Donahue CA - PLoS ONE (2011)

GATA6 Overexpression Correlates with Canonical Wnt Signaling.(A) Wnt signaling activity in AsPC1-GATA6sh and A13A-GATA6sh cells based on TOPFLASH assay. Luciferase activity is represented as the ratio of OT to OF levels in cells with GATA6 knockdown relative to that of mock-transfected cells. (B) Wnt signaling activity in Panc1-GATA6 and HPNE-GATA6 cells determined by TOPFLASH assay. Luciferase activity is represented as the ratio of OT to OF levels in GATA6 transfected cells relative to that of mock-transfected cells. (C and D) Panc1-GATA6 cells were transiently transfected with ß-catenin or mock shRNA and (C) cell proliferation or (D) colony formation determined. All experimental data shown represents the summary three independent experiments. *, p<0.05; **, p<0.01. (E) Immunolabeling patterns of GATA6 and ß-catenin protein in two representative PDAC tissues. Arrows indicate nuclear labeling of both GATA6 and ß-catenin in serial sections of the same cancer tissue. By contrast, the PDAC sample with low GATA6 expression also shows no expression of ß-catenin.
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Related In: Results  -  Collection

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pone-0022129-g004: GATA6 Overexpression Correlates with Canonical Wnt Signaling.(A) Wnt signaling activity in AsPC1-GATA6sh and A13A-GATA6sh cells based on TOPFLASH assay. Luciferase activity is represented as the ratio of OT to OF levels in cells with GATA6 knockdown relative to that of mock-transfected cells. (B) Wnt signaling activity in Panc1-GATA6 and HPNE-GATA6 cells determined by TOPFLASH assay. Luciferase activity is represented as the ratio of OT to OF levels in GATA6 transfected cells relative to that of mock-transfected cells. (C and D) Panc1-GATA6 cells were transiently transfected with ß-catenin or mock shRNA and (C) cell proliferation or (D) colony formation determined. All experimental data shown represents the summary three independent experiments. *, p<0.05; **, p<0.01. (E) Immunolabeling patterns of GATA6 and ß-catenin protein in two representative PDAC tissues. Arrows indicate nuclear labeling of both GATA6 and ß-catenin in serial sections of the same cancer tissue. By contrast, the PDAC sample with low GATA6 expression also shows no expression of ß-catenin.
Mentions: GATA proteins are linked to Wnt signaling in embryogenesis of the heart and lungs [4], [30], [31]. We therefore hypothesized that GATA6 contributes to pancreatic carcinogenesis in part through its effects on Wnt signaling, a putative relationship that has not been explored in any detail for this tumor type. Compared to mock shRNA lentiviral-infected cells, both AsPC1-GATA6sh and A13A-GATA6sh cells showed a significant decrease in functional Wnt signaling activity by TOPFLASH assay (Figure 4A) whereas overexpression of GATA6 in Panc1 and HPNE cells promoted Wnt signaling activity (Figure 4B). To determine if ß-catenin expression is required for these effects we silenced ß-catenin expression using an shRNA strategy in Panc1-GATA6 cells, leading to a significant inhibition of cell proliferation and colony formation (Figure 4C and 4D). In pancreatic cancer tissues, GATA6 overexpression was significantly correlated with nuclear accumulation of ß-catenin protein (8/12 PDACs with GATA6 overexpression showing ß-catenin nuclear accumulation versus 3/20 without GATA6 overexpression, p = 0.004) (Figure 4E). Thus, GATA6 overexpression in PDAC contributes to cell proliferation and colony formation by enhancing canonical Wnt signaling.

Bottom Line: The effects of GATA6 were found to be due to its ability to bind DNA, as forced overexpression of a DNA-binding mutant of GATA6 had no effects on cell growth in vitro or in vivo, nor did they affect Wnt signaling levels in these same cells.Transient transfection of GATA6, but not mutant GATA6, into cancer cell lines led to decreased DKK1 mRNA expression and secretion of DKK1 protein into culture media.Forced overexpression of DKK1 antagonized the effects of GATA6 on Wnt signaling in pancreatic cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins Medical Institutions, Baltimore, Maryland, United States of America.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease characterized by late diagnosis and treatment resistance. Recurrent genetic alterations in defined genes in association with perturbations of developmental cell signaling pathways have been associated with PDAC development and progression. Here, we show that GATA6 contributes to pancreatic carcinogenesis during the temporal progression of pancreatic intraepithelial neoplasia by virtue of Wnt pathway activation. GATA6 is recurrently amplified by both quantitative-PCR and fluorescent in-situ hybridization in human pancreatic intraepithelial neoplasia and in PDAC tissues, and GATA6 copy number is significantly correlated with overall patient survival. Forced overexpression of GATA6 in cancer cell lines enhanced cell proliferation and colony formation in soft agar in vitro and growth in vivo, as well as increased Wnt signaling. By contrast siRNA mediated knockdown of GATA6 led to corresponding decreases in these same parameters. The effects of GATA6 were found to be due to its ability to bind DNA, as forced overexpression of a DNA-binding mutant of GATA6 had no effects on cell growth in vitro or in vivo, nor did they affect Wnt signaling levels in these same cells. A microarray analysis revealed the Wnt antagonist Dickopf-1 (DKK1) as a dysregulated gene in association with GATA6 knockdown, and direct binding of GATA6 to the DKK1 promoter was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assays. Transient transfection of GATA6, but not mutant GATA6, into cancer cell lines led to decreased DKK1 mRNA expression and secretion of DKK1 protein into culture media. Forced overexpression of DKK1 antagonized the effects of GATA6 on Wnt signaling in pancreatic cancer cells. These findings illustrate that one mechanism by which GATA6 promotes pancreatic carcinogenesis is by virtue of its activation of canonical Wnt signaling via regulation of DKK1.

Show MeSH
Related in: MedlinePlus