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N-myc downstream regulated gene 1 modulates Wnt-β-catenin signalling and pleiotropically suppresses metastasis.

Liu W, Xing F, Iiizumi-Gairani M, Okuda H, Watabe M, Pai SK, Pandey PR, Hirota S, Kobayashi A, Mo YY, Fukuda K, Li Y, Watabe K - EMBO Mol Med (2012)

Bottom Line: Here we demonstrate that the tumour metastasis suppressor gene, NDRG1, interacts with the Wnt receptor, LRP6, followed by blocking of the Wnt signalling, and therefore, orchestrates a cellular network that impairs the metastatic progression of tumour cells.Importantly, restoring NDRG1 expression by a small molecule compound significantly suppressed the capability of otherwise highly metastatic tumour cells to thrive in circulation and distant organs in animal models.In addition, our analysis of clinical cohorts data indicate that Wnt+/NDRG-/LRP+ signature has a strong predictable value for recurrence-free survival of cancer patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Southern Illinois University School of Medicine, Springfield, IL, USA.

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Related in: MedlinePlus

NDRG1 blocks Wnt-β-catenin signalling followed by inhibiting ATF3 expressionA. The expression of ATF3 was examined by qRT-PCR or ATF3 reporter assay in PC3mm (left panel) or MCF7 (right panel) that were transfected with or without the expression plasmid of β-catenin/TCF. Western blot was also performed to confirm the ATF3 expression in the presence of β-catenin/TCF. Values are from three independent experiments in triplicate.B-C. Three-base mutation was introduced into the consensus sequence of β-catenin/TCF binding site on the ATF3 gene promoter of the CAT-reporter plasmid. Wild-type and mutant reporter plasmids were then transfected into PC3mm/Tet or MCF7/Tet cells in the presence or absence of tetracycline (B) or Wnt (C). CAT activity of the cell lysates was assayed and normalized by internal Renilla luciferase activity. Western blot analysis of ATF3 expression was also performed. Values are means ± SD of triplicate measurements.D. ChIP assay for PC3mm/Tet cells treated with or without tetracycline for 48 h. Precipitated DNA was subjected to q-PCR using primers specific for the β-catenin binding consensus sequence on the ATF3 promoter (F1R1) as well as a control primer set (F2R2). The ratio of the precipitated DNA was calculated based on cyclic threshold value for each reaction. Values are means ± SD of triplicate measurements. For all experiment, p values were calculated by two-sided Student's t-test.
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fig02: NDRG1 blocks Wnt-β-catenin signalling followed by inhibiting ATF3 expressionA. The expression of ATF3 was examined by qRT-PCR or ATF3 reporter assay in PC3mm (left panel) or MCF7 (right panel) that were transfected with or without the expression plasmid of β-catenin/TCF. Western blot was also performed to confirm the ATF3 expression in the presence of β-catenin/TCF. Values are from three independent experiments in triplicate.B-C. Three-base mutation was introduced into the consensus sequence of β-catenin/TCF binding site on the ATF3 gene promoter of the CAT-reporter plasmid. Wild-type and mutant reporter plasmids were then transfected into PC3mm/Tet or MCF7/Tet cells in the presence or absence of tetracycline (B) or Wnt (C). CAT activity of the cell lysates was assayed and normalized by internal Renilla luciferase activity. Western blot analysis of ATF3 expression was also performed. Values are means ± SD of triplicate measurements.D. ChIP assay for PC3mm/Tet cells treated with or without tetracycline for 48 h. Precipitated DNA was subjected to q-PCR using primers specific for the β-catenin binding consensus sequence on the ATF3 promoter (F1R1) as well as a control primer set (F2R2). The ratio of the precipitated DNA was calculated based on cyclic threshold value for each reaction. Values are means ± SD of triplicate measurements. For all experiment, p values were calculated by two-sided Student's t-test.

Mentions: Previously, we demonstrated that NDRG1 was able to modulate the metastasis-promoting gene ATF3, and therefore, we further explored a possibility that ATF3 expression was regulated by Wnt signalling. We found that Wnt strongly increased the nuclear expression of ATF3 and that the induction of NDRG1 strongly abrogated this effect (Supporting Information Fig S1H). Consistently, ectopic expression of β-catenin and TCF also significantly promoted the expression of ATF3 at both mRNA and protein levels in prostate and breast cancer cells (Fig 2A). Moreover, a deletion of β-catenin/TCF binding consensus sequence of the ATF3 promoter significantly blocked the suppressive effect of NDRG1 (Fig 2B), suggesting that down-regulation of ATF3 by NDRG1 depends directly on the β-catenin/TCF binding site in the ATF3 promoter. In agreement with this result, we found that Wnt treatment led to a significant increase in the ATF3 promoter activity of wild type but not mutant (Fig 2C), and that Wnt substantially augmented the level of ATF3 protein which was decreased upon induced NDRG1 expression (Fig 3B). In addition, our results of ChIP assay by precipitating β-catenin/TCF-chromatin complex clearly showed that β-catenin/TCF binds to the predicted region of ATF3 promoter, while expression of NDRG1 led to more than 95% decrease in binding efficiency (Fig 2D), which provided direct evidence that NDRG1 suppresses ATF3 expression by blocking Wnt-β-catenin signalling.


N-myc downstream regulated gene 1 modulates Wnt-β-catenin signalling and pleiotropically suppresses metastasis.

Liu W, Xing F, Iiizumi-Gairani M, Okuda H, Watabe M, Pai SK, Pandey PR, Hirota S, Kobayashi A, Mo YY, Fukuda K, Li Y, Watabe K - EMBO Mol Med (2012)

NDRG1 blocks Wnt-β-catenin signalling followed by inhibiting ATF3 expressionA. The expression of ATF3 was examined by qRT-PCR or ATF3 reporter assay in PC3mm (left panel) or MCF7 (right panel) that were transfected with or without the expression plasmid of β-catenin/TCF. Western blot was also performed to confirm the ATF3 expression in the presence of β-catenin/TCF. Values are from three independent experiments in triplicate.B-C. Three-base mutation was introduced into the consensus sequence of β-catenin/TCF binding site on the ATF3 gene promoter of the CAT-reporter plasmid. Wild-type and mutant reporter plasmids were then transfected into PC3mm/Tet or MCF7/Tet cells in the presence or absence of tetracycline (B) or Wnt (C). CAT activity of the cell lysates was assayed and normalized by internal Renilla luciferase activity. Western blot analysis of ATF3 expression was also performed. Values are means ± SD of triplicate measurements.D. ChIP assay for PC3mm/Tet cells treated with or without tetracycline for 48 h. Precipitated DNA was subjected to q-PCR using primers specific for the β-catenin binding consensus sequence on the ATF3 promoter (F1R1) as well as a control primer set (F2R2). The ratio of the precipitated DNA was calculated based on cyclic threshold value for each reaction. Values are means ± SD of triplicate measurements. For all experiment, p values were calculated by two-sided Student's t-test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3306556&req=5

fig02: NDRG1 blocks Wnt-β-catenin signalling followed by inhibiting ATF3 expressionA. The expression of ATF3 was examined by qRT-PCR or ATF3 reporter assay in PC3mm (left panel) or MCF7 (right panel) that were transfected with or without the expression plasmid of β-catenin/TCF. Western blot was also performed to confirm the ATF3 expression in the presence of β-catenin/TCF. Values are from three independent experiments in triplicate.B-C. Three-base mutation was introduced into the consensus sequence of β-catenin/TCF binding site on the ATF3 gene promoter of the CAT-reporter plasmid. Wild-type and mutant reporter plasmids were then transfected into PC3mm/Tet or MCF7/Tet cells in the presence or absence of tetracycline (B) or Wnt (C). CAT activity of the cell lysates was assayed and normalized by internal Renilla luciferase activity. Western blot analysis of ATF3 expression was also performed. Values are means ± SD of triplicate measurements.D. ChIP assay for PC3mm/Tet cells treated with or without tetracycline for 48 h. Precipitated DNA was subjected to q-PCR using primers specific for the β-catenin binding consensus sequence on the ATF3 promoter (F1R1) as well as a control primer set (F2R2). The ratio of the precipitated DNA was calculated based on cyclic threshold value for each reaction. Values are means ± SD of triplicate measurements. For all experiment, p values were calculated by two-sided Student's t-test.
Mentions: Previously, we demonstrated that NDRG1 was able to modulate the metastasis-promoting gene ATF3, and therefore, we further explored a possibility that ATF3 expression was regulated by Wnt signalling. We found that Wnt strongly increased the nuclear expression of ATF3 and that the induction of NDRG1 strongly abrogated this effect (Supporting Information Fig S1H). Consistently, ectopic expression of β-catenin and TCF also significantly promoted the expression of ATF3 at both mRNA and protein levels in prostate and breast cancer cells (Fig 2A). Moreover, a deletion of β-catenin/TCF binding consensus sequence of the ATF3 promoter significantly blocked the suppressive effect of NDRG1 (Fig 2B), suggesting that down-regulation of ATF3 by NDRG1 depends directly on the β-catenin/TCF binding site in the ATF3 promoter. In agreement with this result, we found that Wnt treatment led to a significant increase in the ATF3 promoter activity of wild type but not mutant (Fig 2C), and that Wnt substantially augmented the level of ATF3 protein which was decreased upon induced NDRG1 expression (Fig 3B). In addition, our results of ChIP assay by precipitating β-catenin/TCF-chromatin complex clearly showed that β-catenin/TCF binds to the predicted region of ATF3 promoter, while expression of NDRG1 led to more than 95% decrease in binding efficiency (Fig 2D), which provided direct evidence that NDRG1 suppresses ATF3 expression by blocking Wnt-β-catenin signalling.

Bottom Line: Here we demonstrate that the tumour metastasis suppressor gene, NDRG1, interacts with the Wnt receptor, LRP6, followed by blocking of the Wnt signalling, and therefore, orchestrates a cellular network that impairs the metastatic progression of tumour cells.Importantly, restoring NDRG1 expression by a small molecule compound significantly suppressed the capability of otherwise highly metastatic tumour cells to thrive in circulation and distant organs in animal models.In addition, our analysis of clinical cohorts data indicate that Wnt+/NDRG-/LRP+ signature has a strong predictable value for recurrence-free survival of cancer patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Southern Illinois University School of Medicine, Springfield, IL, USA.

Show MeSH
Related in: MedlinePlus