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MUC1 regulates cyclin D1 gene expression through p120 catenin and β-catenin.

Liu X, Caffrey TC, Steele MM, Mohr A, Singh PK, Radhakrishnan P, Kelly DL, Wen Y, Hollingsworth MA - Oncogenesis (2014)

Bottom Line: We observed a significant enhancement in the activation of cyclin D1 promoter-reporter activity in poorly differentiated Panc1.MUC1F cells that overexpress recombinant MUC1 relative to Panc-1.NEO cells, which express very low levels of endogenous MUC1.Further, MUC1 affected subcellular localization of p120 catenin in association with one of the main effectors of P120 catenin, the transcriptional repressor Kaiso, supporting the hypothesis that p120 catenin relieved transcriptional repression by Kaiso.Our data show MUC1 enhances the activities of both β-catenin and p120 catenin.

View Article: PubMed Central - PubMed

Affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA.

ABSTRACT
MUC1 interacts with β-catenin and p120 catenin to modulate WNT signaling. We investigated the effect of overexpressing MUC1 on the regulation of cyclin D1, a downstream target for the WNT/β-catenin signaling pathway, in two human pancreatic cancer cell lines, Panc-1 and S2-013. We observed a significant enhancement in the activation of cyclin D1 promoter-reporter activity in poorly differentiated Panc1.MUC1F cells that overexpress recombinant MUC1 relative to Panc-1.NEO cells, which express very low levels of endogenous MUC1. In stark contrast, cyclin D1 promoter activity was not affected in moderately differentiated S2-013.MUC1F cells that overexpressed recombinant MUC1 relative to S2-013.NEO cells that expressed low levels of endogenous MUC1. The S2-013 cell line was recently shown to be deficient in p120 catenin. MUC1 is known to interact with P120 catenin. We show here that re-expression of different isoforms of p120 catenin restored cyclin D1 promoter activity. Further, MUC1 affected subcellular localization of p120 catenin in association with one of the main effectors of P120 catenin, the transcriptional repressor Kaiso, supporting the hypothesis that p120 catenin relieved transcriptional repression by Kaiso. Thus, full activation of cyclin D1 promoter activity requires β-catenin activation of TCF-lef and stabilization of specific p120 catenin isoforms to relieve the repression of KAISO. Our data show MUC1 enhances the activities of both β-catenin and p120 catenin.

No MeSH data available.


Related in: MedlinePlus

Luciferase assay detecting effect of MUC1 on cyclin D1 promoter activity. Cells were transfected with 300 or 600 ng of the reporter plasmids containing 1745 bp of the cyclin D1 gene promoter (−1745CD1Luc, TOPFLASH) consists of wild-type LEF-1/Tcf-4-binding site or reporter plasmids containing mutated LEF-1/Tcf-4-binding site in the cyclin D1 gene promoter (FOPFLASH), together with 400 ng of a synthetic Renilla luciferase reporter plasmids SV40 as transfection control. Each transfection was carried out in duplicate plates, and the data (relative lumenescence units-RLU) shown here are representative of three independent experiments. (a) Panc-1.MUC1F or Panc-1.Neo (95% confluence). (b) S2-013.MUC1F cells, S2-013.NEO cells, S2-013 GFP-NEO, two clones of S2-013 cells in which MUC1 was knocked down by MUC1-siRNA—S2-013.MTII.C1 and S2-013.MTII.C2 (95% confluence).
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fig1: Luciferase assay detecting effect of MUC1 on cyclin D1 promoter activity. Cells were transfected with 300 or 600 ng of the reporter plasmids containing 1745 bp of the cyclin D1 gene promoter (−1745CD1Luc, TOPFLASH) consists of wild-type LEF-1/Tcf-4-binding site or reporter plasmids containing mutated LEF-1/Tcf-4-binding site in the cyclin D1 gene promoter (FOPFLASH), together with 400 ng of a synthetic Renilla luciferase reporter plasmids SV40 as transfection control. Each transfection was carried out in duplicate plates, and the data (relative lumenescence units-RLU) shown here are representative of three independent experiments. (a) Panc-1.MUC1F or Panc-1.Neo (95% confluence). (b) S2-013.MUC1F cells, S2-013.NEO cells, S2-013 GFP-NEO, two clones of S2-013 cells in which MUC1 was knocked down by MUC1-siRNA—S2-013.MTII.C1 and S2-013.MTII.C2 (95% confluence).

Mentions: There was no activity of cyclin D1 promoters with mutated LEF/Tcf-4-binding sites in NEO control and MUC1-expressing cells (Figure 1). In contrast, for the construct containing wild-type sequences, there was low cyclin D1 promoter activity in NEO control cells, but there was a significant enhancement (approximately 100-fold) for cyclin D1 promoter activity in Panc-1.MUC1F (Figure 1). This supported the hypothesis that WNT signaling through β-catenin-activated TCF/LEF in these cells and that MUC1 stabilization of β-catenin dramatically increased cyclin D1 promoter activity. Curiously, however, we found that cyclin D1 promoter activity in S2-013 cells was low and unresponsive to alterations in MUC1 expression (Figure 1), either in S2-013 cells expressing high levels of MUC1 or in cells in which the low levels of endogenous MUC1 was knocked down by RNA interference. This negative result indicated that MUC1 stabilization of β-catenin is not sufficient to activate or influence cyclin D1 promoter activity and that transactivation of the cyclin D1 gene promoter in this cell line is affected by other factors.


MUC1 regulates cyclin D1 gene expression through p120 catenin and β-catenin.

Liu X, Caffrey TC, Steele MM, Mohr A, Singh PK, Radhakrishnan P, Kelly DL, Wen Y, Hollingsworth MA - Oncogenesis (2014)

Luciferase assay detecting effect of MUC1 on cyclin D1 promoter activity. Cells were transfected with 300 or 600 ng of the reporter plasmids containing 1745 bp of the cyclin D1 gene promoter (−1745CD1Luc, TOPFLASH) consists of wild-type LEF-1/Tcf-4-binding site or reporter plasmids containing mutated LEF-1/Tcf-4-binding site in the cyclin D1 gene promoter (FOPFLASH), together with 400 ng of a synthetic Renilla luciferase reporter plasmids SV40 as transfection control. Each transfection was carried out in duplicate plates, and the data (relative lumenescence units-RLU) shown here are representative of three independent experiments. (a) Panc-1.MUC1F or Panc-1.Neo (95% confluence). (b) S2-013.MUC1F cells, S2-013.NEO cells, S2-013 GFP-NEO, two clones of S2-013 cells in which MUC1 was knocked down by MUC1-siRNA—S2-013.MTII.C1 and S2-013.MTII.C2 (95% confluence).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150213&req=5

fig1: Luciferase assay detecting effect of MUC1 on cyclin D1 promoter activity. Cells were transfected with 300 or 600 ng of the reporter plasmids containing 1745 bp of the cyclin D1 gene promoter (−1745CD1Luc, TOPFLASH) consists of wild-type LEF-1/Tcf-4-binding site or reporter plasmids containing mutated LEF-1/Tcf-4-binding site in the cyclin D1 gene promoter (FOPFLASH), together with 400 ng of a synthetic Renilla luciferase reporter plasmids SV40 as transfection control. Each transfection was carried out in duplicate plates, and the data (relative lumenescence units-RLU) shown here are representative of three independent experiments. (a) Panc-1.MUC1F or Panc-1.Neo (95% confluence). (b) S2-013.MUC1F cells, S2-013.NEO cells, S2-013 GFP-NEO, two clones of S2-013 cells in which MUC1 was knocked down by MUC1-siRNA—S2-013.MTII.C1 and S2-013.MTII.C2 (95% confluence).
Mentions: There was no activity of cyclin D1 promoters with mutated LEF/Tcf-4-binding sites in NEO control and MUC1-expressing cells (Figure 1). In contrast, for the construct containing wild-type sequences, there was low cyclin D1 promoter activity in NEO control cells, but there was a significant enhancement (approximately 100-fold) for cyclin D1 promoter activity in Panc-1.MUC1F (Figure 1). This supported the hypothesis that WNT signaling through β-catenin-activated TCF/LEF in these cells and that MUC1 stabilization of β-catenin dramatically increased cyclin D1 promoter activity. Curiously, however, we found that cyclin D1 promoter activity in S2-013 cells was low and unresponsive to alterations in MUC1 expression (Figure 1), either in S2-013 cells expressing high levels of MUC1 or in cells in which the low levels of endogenous MUC1 was knocked down by RNA interference. This negative result indicated that MUC1 stabilization of β-catenin is not sufficient to activate or influence cyclin D1 promoter activity and that transactivation of the cyclin D1 gene promoter in this cell line is affected by other factors.

Bottom Line: We observed a significant enhancement in the activation of cyclin D1 promoter-reporter activity in poorly differentiated Panc1.MUC1F cells that overexpress recombinant MUC1 relative to Panc-1.NEO cells, which express very low levels of endogenous MUC1.Further, MUC1 affected subcellular localization of p120 catenin in association with one of the main effectors of P120 catenin, the transcriptional repressor Kaiso, supporting the hypothesis that p120 catenin relieved transcriptional repression by Kaiso.Our data show MUC1 enhances the activities of both β-catenin and p120 catenin.

View Article: PubMed Central - PubMed

Affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA.

ABSTRACT
MUC1 interacts with β-catenin and p120 catenin to modulate WNT signaling. We investigated the effect of overexpressing MUC1 on the regulation of cyclin D1, a downstream target for the WNT/β-catenin signaling pathway, in two human pancreatic cancer cell lines, Panc-1 and S2-013. We observed a significant enhancement in the activation of cyclin D1 promoter-reporter activity in poorly differentiated Panc1.MUC1F cells that overexpress recombinant MUC1 relative to Panc-1.NEO cells, which express very low levels of endogenous MUC1. In stark contrast, cyclin D1 promoter activity was not affected in moderately differentiated S2-013.MUC1F cells that overexpressed recombinant MUC1 relative to S2-013.NEO cells that expressed low levels of endogenous MUC1. The S2-013 cell line was recently shown to be deficient in p120 catenin. MUC1 is known to interact with P120 catenin. We show here that re-expression of different isoforms of p120 catenin restored cyclin D1 promoter activity. Further, MUC1 affected subcellular localization of p120 catenin in association with one of the main effectors of P120 catenin, the transcriptional repressor Kaiso, supporting the hypothesis that p120 catenin relieved transcriptional repression by Kaiso. Thus, full activation of cyclin D1 promoter activity requires β-catenin activation of TCF-lef and stabilization of specific p120 catenin isoforms to relieve the repression of KAISO. Our data show MUC1 enhances the activities of both β-catenin and p120 catenin.

No MeSH data available.


Related in: MedlinePlus