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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 Copy Number Gain Correlates with Intraductal Progression of Pancreatic Cancer.(A) GATA6 copy number (mean ± SE) in microdissected normal ducts (N = 4), PanIN-1 (N = 13), PanIN-2 (10), PanIN-3 (N = 17) lesions and pancreatic cancer (N = 55). (B) Representative FISH of the nucleus of a neoplastic cell within a PanIN3 lesion with >11-fold GATA6 amplification (right) compared to the nucleus of a neoplastic cell from a different PanIN3 lesion without copy number gain of GATA6 (left). GATA6 probe was labeled with red and chromosome 18 centromere probe (18 Cent) was labeled with green. The sections were counterstained with DAPI to highlight nuclei. (C) Correlation of GATA6 mRNA expression and copy number in microdissected samples of normal, PanIN and cancer tissue. (D) GATA6 immunolabeling of two pancreatic cancer tissues with GATA6 copy number gain compared to two cancers without copy number gain. Increased copy number is highly associated with nuclear labeling of GATA6 protein. (E) Kaplan Meier survival curve illustrating the relationship of GATA6 copy number gain (≥2.3 copies per haploid genome) to overall survival in patients with surgically resected pancreatic cancer.
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pone-0022129-g001: GATA6 Copy Number Gain Correlates with Intraductal Progression of Pancreatic Cancer.(A) GATA6 copy number (mean ± SE) in microdissected normal ducts (N = 4), PanIN-1 (N = 13), PanIN-2 (10), PanIN-3 (N = 17) lesions and pancreatic cancer (N = 55). (B) Representative FISH of the nucleus of a neoplastic cell within a PanIN3 lesion with >11-fold GATA6 amplification (right) compared to the nucleus of a neoplastic cell from a different PanIN3 lesion without copy number gain of GATA6 (left). GATA6 probe was labeled with red and chromosome 18 centromere probe (18 Cent) was labeled with green. The sections were counterstained with DAPI to highlight nuclei. (C) Correlation of GATA6 mRNA expression and copy number in microdissected samples of normal, PanIN and cancer tissue. (D) GATA6 immunolabeling of two pancreatic cancer tissues with GATA6 copy number gain compared to two cancers without copy number gain. Increased copy number is highly associated with nuclear labeling of GATA6 protein. (E) Kaplan Meier survival curve illustrating the relationship of GATA6 copy number gain (≥2.3 copies per haploid genome) to overall survival in patients with surgically resected pancreatic cancer.

Mentions: Normal pancreatic ductal epithelium is believed to progress to infiltrating cancer through a series of morphologically defined precursors called pancreatic intraepithelial neoplasia (PanIN-1, 2, 3) [27]. To understand the correlation between genetic gain of GATA6 and PDAC development, we assessed GATA6 copy number in microdissected samples of normal duct epithelium, PanIN, and human PDAC by quantitative PCR. Relative to the haploid genome, there was no gain of GATA6 in normal duct epithelium (0 of 4), PanIN-1 (0 of 13) or PanIN-2 (0 of 10) lesions. By contrast, increased GATA6 copy number (≥2.3 copies) was identified in 6/17 samples (35%) of PanIN-3 and in 18/55 samples (33%) of PDAC (Figure 1A). GATA6 copy number gain was further confirmed by fluorescent in situ hybridization (FISH) in paraffin-embedded sections of one PanIN-3 and 10 PDAC samples (Figure 1B).


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 Copy Number Gain Correlates with Intraductal Progression of Pancreatic Cancer.(A) GATA6 copy number (mean ± SE) in microdissected normal ducts (N = 4), PanIN-1 (N = 13), PanIN-2 (10), PanIN-3 (N = 17) lesions and pancreatic cancer (N = 55). (B) Representative FISH of the nucleus of a neoplastic cell within a PanIN3 lesion with >11-fold GATA6 amplification (right) compared to the nucleus of a neoplastic cell from a different PanIN3 lesion without copy number gain of GATA6 (left). GATA6 probe was labeled with red and chromosome 18 centromere probe (18 Cent) was labeled with green. The sections were counterstained with DAPI to highlight nuclei. (C) Correlation of GATA6 mRNA expression and copy number in microdissected samples of normal, PanIN and cancer tissue. (D) GATA6 immunolabeling of two pancreatic cancer tissues with GATA6 copy number gain compared to two cancers without copy number gain. Increased copy number is highly associated with nuclear labeling of GATA6 protein. (E) Kaplan Meier survival curve illustrating the relationship of GATA6 copy number gain (≥2.3 copies per haploid genome) to overall survival in patients with surgically resected pancreatic cancer.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3139620&req=5

pone-0022129-g001: GATA6 Copy Number Gain Correlates with Intraductal Progression of Pancreatic Cancer.(A) GATA6 copy number (mean ± SE) in microdissected normal ducts (N = 4), PanIN-1 (N = 13), PanIN-2 (10), PanIN-3 (N = 17) lesions and pancreatic cancer (N = 55). (B) Representative FISH of the nucleus of a neoplastic cell within a PanIN3 lesion with >11-fold GATA6 amplification (right) compared to the nucleus of a neoplastic cell from a different PanIN3 lesion without copy number gain of GATA6 (left). GATA6 probe was labeled with red and chromosome 18 centromere probe (18 Cent) was labeled with green. The sections were counterstained with DAPI to highlight nuclei. (C) Correlation of GATA6 mRNA expression and copy number in microdissected samples of normal, PanIN and cancer tissue. (D) GATA6 immunolabeling of two pancreatic cancer tissues with GATA6 copy number gain compared to two cancers without copy number gain. Increased copy number is highly associated with nuclear labeling of GATA6 protein. (E) Kaplan Meier survival curve illustrating the relationship of GATA6 copy number gain (≥2.3 copies per haploid genome) to overall survival in patients with surgically resected pancreatic cancer.
Mentions: Normal pancreatic ductal epithelium is believed to progress to infiltrating cancer through a series of morphologically defined precursors called pancreatic intraepithelial neoplasia (PanIN-1, 2, 3) [27]. To understand the correlation between genetic gain of GATA6 and PDAC development, we assessed GATA6 copy number in microdissected samples of normal duct epithelium, PanIN, and human PDAC by quantitative PCR. Relative to the haploid genome, there was no gain of GATA6 in normal duct epithelium (0 of 4), PanIN-1 (0 of 13) or PanIN-2 (0 of 10) lesions. By contrast, increased GATA6 copy number (≥2.3 copies) was identified in 6/17 samples (35%) of PanIN-3 and in 18/55 samples (33%) of PDAC (Figure 1A). GATA6 copy number gain was further confirmed by fluorescent in situ hybridization (FISH) in paraffin-embedded sections of one PanIN-3 and 10 PDAC samples (Figure 1B).

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