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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

Western blot protein levels of cyclin D1 in pancreatic cancer cell lines. (a, b) Membrane/cytoplasmic extracts and nuclear extracts from Panc-1.MUC1F, Panc-1.NEO, S2-013.MUC1F, S2-013.NEO, S2-013 GFP-NEO, two clones of MUC1-siRNA S2-013 cells were subjected to 4–20% SDS–PAGE and analyzed by immunoblot (IB) with an anti-cyclin D1 mAb and β-actin as a loading control. Right panel in a shows densitometry analysis of the signal for nuclear cyclin D1 normalized to the signal for β-actin. *P<0.01, significant difference. (c) Cyclin D1 protein expression is enhanced by expression of MUC1 in Panc-1.MUC1F cells as compared with Panc-1.NEO cells. Confocal microscopy was used to determine the cyclin D1 protein expression in Panc-1.MUC1F and Panc-1.NEO cells. Cells grown above 90% confluence on coverslips were fixed with 4% paraformaldehyde and permeablized with Trixton X-100 before incubation with mAb anti-cyclin D1, which was identified with fluorescein isothiocyanate-conjugated secondary antibody and visualized as green color. Images were examined with a Zeiss LSM 410 laser scanning microscope (Bar=20 μM; × 100 magnification; results shown here represent three or four individual cell scanning observations.).
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fig2: Western blot protein levels of cyclin D1 in pancreatic cancer cell lines. (a, b) Membrane/cytoplasmic extracts and nuclear extracts from Panc-1.MUC1F, Panc-1.NEO, S2-013.MUC1F, S2-013.NEO, S2-013 GFP-NEO, two clones of MUC1-siRNA S2-013 cells were subjected to 4–20% SDS–PAGE and analyzed by immunoblot (IB) with an anti-cyclin D1 mAb and β-actin as a loading control. Right panel in a shows densitometry analysis of the signal for nuclear cyclin D1 normalized to the signal for β-actin. *P<0.01, significant difference. (c) Cyclin D1 protein expression is enhanced by expression of MUC1 in Panc-1.MUC1F cells as compared with Panc-1.NEO cells. Confocal microscopy was used to determine the cyclin D1 protein expression in Panc-1.MUC1F and Panc-1.NEO cells. Cells grown above 90% confluence on coverslips were fixed with 4% paraformaldehyde and permeablized with Trixton X-100 before incubation with mAb anti-cyclin D1, which was identified with fluorescein isothiocyanate-conjugated secondary antibody and visualized as green color. Images were examined with a Zeiss LSM 410 laser scanning microscope (Bar=20 μM; × 100 magnification; results shown here represent three or four individual cell scanning observations.).

Mentions: We confirmed the consequences of expressing MUC1 on steady-state levels of endogenous cyclin D1 in the Panc1 and S2-013 pancreatic cancer cell lines. Consistent with the promoter-reporter assay results, expression of MUC1 elevated the steady-state level of cyclin D1 transcripts (data not shown) and cyclin D1 protein in Panc-1 cells, but not in S2-013 cells (Figure 2). These results suggest that expression of MUC1 contributes to the increase in cyclin D1 transcripts in the Panc-1 cells, but not in S2-013 cells. Previous studies reported that expression of MUC1 co-activated the cyclin D1 gene by transactivation in human HCT116 colon carcinoma cells.24 A recent report suggests that MUC1 induces TCF7L2 transcription factor activation and promotes cyclin D1 expression in human breast cancer cells.22 Taken together, these results indicate that the effects of MUC1 expression on cyclin D1 gene expression vary among different cell types.


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)

Western blot protein levels of cyclin D1 in pancreatic cancer cell lines. (a, b) Membrane/cytoplasmic extracts and nuclear extracts from Panc-1.MUC1F, Panc-1.NEO, S2-013.MUC1F, S2-013.NEO, S2-013 GFP-NEO, two clones of MUC1-siRNA S2-013 cells were subjected to 4–20% SDS–PAGE and analyzed by immunoblot (IB) with an anti-cyclin D1 mAb and β-actin as a loading control. Right panel in a shows densitometry analysis of the signal for nuclear cyclin D1 normalized to the signal for β-actin. *P<0.01, significant difference. (c) Cyclin D1 protein expression is enhanced by expression of MUC1 in Panc-1.MUC1F cells as compared with Panc-1.NEO cells. Confocal microscopy was used to determine the cyclin D1 protein expression in Panc-1.MUC1F and Panc-1.NEO cells. Cells grown above 90% confluence on coverslips were fixed with 4% paraformaldehyde and permeablized with Trixton X-100 before incubation with mAb anti-cyclin D1, which was identified with fluorescein isothiocyanate-conjugated secondary antibody and visualized as green color. Images were examined with a Zeiss LSM 410 laser scanning microscope (Bar=20 μM; × 100 magnification; results shown here represent three or four individual cell scanning observations.).
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Related In: Results  -  Collection

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fig2: Western blot protein levels of cyclin D1 in pancreatic cancer cell lines. (a, b) Membrane/cytoplasmic extracts and nuclear extracts from Panc-1.MUC1F, Panc-1.NEO, S2-013.MUC1F, S2-013.NEO, S2-013 GFP-NEO, two clones of MUC1-siRNA S2-013 cells were subjected to 4–20% SDS–PAGE and analyzed by immunoblot (IB) with an anti-cyclin D1 mAb and β-actin as a loading control. Right panel in a shows densitometry analysis of the signal for nuclear cyclin D1 normalized to the signal for β-actin. *P<0.01, significant difference. (c) Cyclin D1 protein expression is enhanced by expression of MUC1 in Panc-1.MUC1F cells as compared with Panc-1.NEO cells. Confocal microscopy was used to determine the cyclin D1 protein expression in Panc-1.MUC1F and Panc-1.NEO cells. Cells grown above 90% confluence on coverslips were fixed with 4% paraformaldehyde and permeablized with Trixton X-100 before incubation with mAb anti-cyclin D1, which was identified with fluorescein isothiocyanate-conjugated secondary antibody and visualized as green color. Images were examined with a Zeiss LSM 410 laser scanning microscope (Bar=20 μM; × 100 magnification; results shown here represent three or four individual cell scanning observations.).
Mentions: We confirmed the consequences of expressing MUC1 on steady-state levels of endogenous cyclin D1 in the Panc1 and S2-013 pancreatic cancer cell lines. Consistent with the promoter-reporter assay results, expression of MUC1 elevated the steady-state level of cyclin D1 transcripts (data not shown) and cyclin D1 protein in Panc-1 cells, but not in S2-013 cells (Figure 2). These results suggest that expression of MUC1 contributes to the increase in cyclin D1 transcripts in the Panc-1 cells, but not in S2-013 cells. Previous studies reported that expression of MUC1 co-activated the cyclin D1 gene by transactivation in human HCT116 colon carcinoma cells.24 A recent report suggests that MUC1 induces TCF7L2 transcription factor activation and promotes cyclin D1 expression in human breast cancer cells.22 Taken together, these results indicate that the effects of MUC1 expression on cyclin D1 gene expression vary among different cell types.

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