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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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Effects of GATA6 Knockdown on Cell Growth in vitro and in vivo.(A) Total protein was extracted from AsPC1-GATA6sh and A13A-GATA6sh cells and mock shRNA controls and analyzed by Western blot for relative levels of GATA6 protein relative to actin. (B) Real-time PCR for GATA6 expression in AsPC1-GATA6sh and A13A-GATA6sh cells. (C) These cells were also analyzed for cell proliferation at different time points, (D) cultured in soft agar and the number of colonies at 2 weeks counted, and (E) analyzed by flow cytometry to determine the percent of cells in G2/M phase. (F) Representative xenograft formation in vivo (above) and after explantation (lower) of AsPC1 control and GATA6sh cells at 8 weeks postinjection. (G) Average tumor volume (mean ± SE) of these same xenografts at 8 weeks postinjection. Similar results were noted for A13A-GATA6sh cells (data not shown). With exception of flow cytometry that was performed in duplicate, all experimental data shown represents the summary three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.
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pone-0022129-g002: Effects of GATA6 Knockdown on Cell Growth in vitro and in vivo.(A) Total protein was extracted from AsPC1-GATA6sh and A13A-GATA6sh cells and mock shRNA controls and analyzed by Western blot for relative levels of GATA6 protein relative to actin. (B) Real-time PCR for GATA6 expression in AsPC1-GATA6sh and A13A-GATA6sh cells. (C) These cells were also analyzed for cell proliferation at different time points, (D) cultured in soft agar and the number of colonies at 2 weeks counted, and (E) analyzed by flow cytometry to determine the percent of cells in G2/M phase. (F) Representative xenograft formation in vivo (above) and after explantation (lower) of AsPC1 control and GATA6sh cells at 8 weeks postinjection. (G) Average tumor volume (mean ± SE) of these same xenografts at 8 weeks postinjection. Similar results were noted for A13A-GATA6sh cells (data not shown). With exception of flow cytometry that was performed in duplicate, all experimental data shown represents the summary three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.

Mentions: Amplification and overexpression of GATA6 in PanIN and PDAC suggests it contributes to PDAC biology [8], [9]. We therefore constructed lentiviral vectors that express either a mock or GATA6 specific shRNA and used them to stably infect the PDAC cell lines AsPC1 and A13A with copy numbers of 2.3 and 9.0 relative to the haploid genome, respectively [8], [9]. In both cell lines, GATA6 is also overexpressed at least 10-fold relative to normal duct cells [8], [9] (Figure S2). Knockdown of GATA6 in both cell lines (Figure 2A, 2B) led to significant decreases in cell proliferation and colony formation (Figure 2C and D), a reduction in cells within G2/M phase (Figure 2E) and decreased growth in vivo (Figure 2F and 2G). Conversely, forced overexpression of GATA6 in the PDAC cell line Panc1 with low levels of endogenous GATA6 expression [8] (Figure 3A and Figure S2) led to increased cell proliferation and colony formation (Figure 3B and 3C) similar to that also previously shown for the cell line MiaPaca2 that does not have endogenous expression of GATA6 [8].


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)

Effects of GATA6 Knockdown on Cell Growth in vitro and in vivo.(A) Total protein was extracted from AsPC1-GATA6sh and A13A-GATA6sh cells and mock shRNA controls and analyzed by Western blot for relative levels of GATA6 protein relative to actin. (B) Real-time PCR for GATA6 expression in AsPC1-GATA6sh and A13A-GATA6sh cells. (C) These cells were also analyzed for cell proliferation at different time points, (D) cultured in soft agar and the number of colonies at 2 weeks counted, and (E) analyzed by flow cytometry to determine the percent of cells in G2/M phase. (F) Representative xenograft formation in vivo (above) and after explantation (lower) of AsPC1 control and GATA6sh cells at 8 weeks postinjection. (G) Average tumor volume (mean ± SE) of these same xenografts at 8 weeks postinjection. Similar results were noted for A13A-GATA6sh cells (data not shown). With exception of flow cytometry that was performed in duplicate, all experimental data shown represents the summary three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3139620&req=5

pone-0022129-g002: Effects of GATA6 Knockdown on Cell Growth in vitro and in vivo.(A) Total protein was extracted from AsPC1-GATA6sh and A13A-GATA6sh cells and mock shRNA controls and analyzed by Western blot for relative levels of GATA6 protein relative to actin. (B) Real-time PCR for GATA6 expression in AsPC1-GATA6sh and A13A-GATA6sh cells. (C) These cells were also analyzed for cell proliferation at different time points, (D) cultured in soft agar and the number of colonies at 2 weeks counted, and (E) analyzed by flow cytometry to determine the percent of cells in G2/M phase. (F) Representative xenograft formation in vivo (above) and after explantation (lower) of AsPC1 control and GATA6sh cells at 8 weeks postinjection. (G) Average tumor volume (mean ± SE) of these same xenografts at 8 weeks postinjection. Similar results were noted for A13A-GATA6sh cells (data not shown). With exception of flow cytometry that was performed in duplicate, all experimental data shown represents the summary three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.
Mentions: Amplification and overexpression of GATA6 in PanIN and PDAC suggests it contributes to PDAC biology [8], [9]. We therefore constructed lentiviral vectors that express either a mock or GATA6 specific shRNA and used them to stably infect the PDAC cell lines AsPC1 and A13A with copy numbers of 2.3 and 9.0 relative to the haploid genome, respectively [8], [9]. In both cell lines, GATA6 is also overexpressed at least 10-fold relative to normal duct cells [8], [9] (Figure S2). Knockdown of GATA6 in both cell lines (Figure 2A, 2B) led to significant decreases in cell proliferation and colony formation (Figure 2C and D), a reduction in cells within G2/M phase (Figure 2E) and decreased growth in vivo (Figure 2F and 2G). Conversely, forced overexpression of GATA6 in the PDAC cell line Panc1 with low levels of endogenous GATA6 expression [8] (Figure 3A and Figure S2) led to increased cell proliferation and colony formation (Figure 3B and 3C) similar to that also previously shown for the cell line MiaPaca2 that does not have endogenous expression of GATA6 [8].

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