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MUC4 is negatively regulated through the Wnt/ β -catenin pathway via the Notch effector Hath1 in colorectal cancer

View Article: PubMed Central - PubMed

ABSTRACT

MUC4 is a transmembrane mucin lining the normal colonic epithelium. The aberrant/de novo over-expression of MUC4 is well documented in malignancies of the pancreas, ovary and breast. However, studies have reported the loss of MUC4 expression in the majority of colorectal cancers (CRCs). A MUC4 promoter analysis showed the presence of three putative TCF/LEF sites, implying a possible regulation by the Wnt/β-catenin pathway, which has been shown to drive CRC progression. Thus, the objective of our study was to determine whether MUC4 is regulated by β-catenin in CRC. We first knocked down (KD) β-catenin in three CRC cell lines; LS180, HCT-8 and HCT116, which resulted in increased MUC4 transcript and MUC4 protein. Additionally, the overexpression of stabilized mutant β-catenin in LS180 and HCT-8 resulted in a decrease in MUC4 expression. Immunohistochemistry (IHC) of mouse colon tissue harboring tubular adenomas and high grade dysplasia showed dramatically reduced Muc4 in lesions relative to adjacent normal tissue, with increased cytosolic/nuclear β-catenin. Luciferase assays with the complete MUC4 promoter construct p3778 showed increased MUC4 promoter luciferase activity in the absence of β-catenin (KD). Mutation of all three putative TCF/LEF sites showed that MUC4 promoter luciferase activity was increased relative to the un-mutated promoter. Interestingly, it was observed that MUC4 expressing CRC cell lines also expressed high levels of Hath1, a transcription factor repressed by both active Wnt/β-catenin and Notch signaling. The KD of β-catenin and/or treatment with a Notch γ-secretase inhibitor, Dibenzazepine (DBZ) resulted in increased Hath1 and MUC4 in LS180, HCT-8 and HCT116. Furthermore, overexpression of Hath1 in HCT-8 and LS180 caused increased MUC4 transcript and MUC4 protein. Taken together, our results indicate that the Wnt/β-catenin pathway suppresses the Notch pathway effector Hath1, resulting in reduced MUC4 in CRC.

No MeSH data available.


Increased nuclear β-catenin is associated with reduced MUC4 expression(A) A panel of CRC cell lines was profiled for the expression of MUC4 and β-catenin. β-actin was used as a loading control. (B) Confocal microscopy with MUC4 (green) antibody shows that HCT-8 cells express MUC4 abundantly while HCT116 cells show very low MUC4 expression. β-catenin (red) is present in both cell lines.(C) Immunohistochemical staining for mouse Muc4 and β-catenin in colon sections from ApcMin/Cldn-1 mice treated with DSS (left panel), ApcMin mice treated with DSS (middle) and ApcMin given water (right panel). Staining for β-catenin (upper panel) and Muc4 (lower panel) showed intense cytosolic/nuclear staining for β-catenin and depletion of Muc4 in lesions (solid arrow), while surrounding normal areas showed reduced β-catenin and intense goblet cell staining for Muc4 (dotted arrow). Table shows type, number of lesions in in mice either treated with DSS alone or ApcMin mice treated with DSS.
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Figure 1: Increased nuclear β-catenin is associated with reduced MUC4 expression(A) A panel of CRC cell lines was profiled for the expression of MUC4 and β-catenin. β-actin was used as a loading control. (B) Confocal microscopy with MUC4 (green) antibody shows that HCT-8 cells express MUC4 abundantly while HCT116 cells show very low MUC4 expression. β-catenin (red) is present in both cell lines.(C) Immunohistochemical staining for mouse Muc4 and β-catenin in colon sections from ApcMin/Cldn-1 mice treated with DSS (left panel), ApcMin mice treated with DSS (middle) and ApcMin given water (right panel). Staining for β-catenin (upper panel) and Muc4 (lower panel) showed intense cytosolic/nuclear staining for β-catenin and depletion of Muc4 in lesions (solid arrow), while surrounding normal areas showed reduced β-catenin and intense goblet cell staining for Muc4 (dotted arrow). Table shows type, number of lesions in in mice either treated with DSS alone or ApcMin mice treated with DSS.

Mentions: In order to determine whether aberrant β-catenin expression/localization correlated with MUC4 expression in CRC, we first examined the expression of MUC4 and β-catenin in CRC cell lines. While all cell lines examined expressed β-catenin, only two of the 7 cell lines were found to express MUC4 abundantly; LS180 and HCT-8 (Figure 1A), while MUC4 expression in HCT116 was negligible to none. However, with confocal microscopy, HCT116 was observed to express low levels of MUC4, in contrast with the HCT-8 cell line, where MUC4 was abundantly expressed (Figure 1B). Thus, a majority of the CRC cell lines examined displayed a loss of MUC4.


MUC4 is negatively regulated through the Wnt/ β -catenin pathway via the Notch effector Hath1 in colorectal cancer
Increased nuclear β-catenin is associated with reduced MUC4 expression(A) A panel of CRC cell lines was profiled for the expression of MUC4 and β-catenin. β-actin was used as a loading control. (B) Confocal microscopy with MUC4 (green) antibody shows that HCT-8 cells express MUC4 abundantly while HCT116 cells show very low MUC4 expression. β-catenin (red) is present in both cell lines.(C) Immunohistochemical staining for mouse Muc4 and β-catenin in colon sections from ApcMin/Cldn-1 mice treated with DSS (left panel), ApcMin mice treated with DSS (middle) and ApcMin given water (right panel). Staining for β-catenin (upper panel) and Muc4 (lower panel) showed intense cytosolic/nuclear staining for β-catenin and depletion of Muc4 in lesions (solid arrow), while surrounding normal areas showed reduced β-catenin and intense goblet cell staining for Muc4 (dotted arrow). Table shows type, number of lesions in in mice either treated with DSS alone or ApcMin mice treated with DSS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4979589&req=5

Figure 1: Increased nuclear β-catenin is associated with reduced MUC4 expression(A) A panel of CRC cell lines was profiled for the expression of MUC4 and β-catenin. β-actin was used as a loading control. (B) Confocal microscopy with MUC4 (green) antibody shows that HCT-8 cells express MUC4 abundantly while HCT116 cells show very low MUC4 expression. β-catenin (red) is present in both cell lines.(C) Immunohistochemical staining for mouse Muc4 and β-catenin in colon sections from ApcMin/Cldn-1 mice treated with DSS (left panel), ApcMin mice treated with DSS (middle) and ApcMin given water (right panel). Staining for β-catenin (upper panel) and Muc4 (lower panel) showed intense cytosolic/nuclear staining for β-catenin and depletion of Muc4 in lesions (solid arrow), while surrounding normal areas showed reduced β-catenin and intense goblet cell staining for Muc4 (dotted arrow). Table shows type, number of lesions in in mice either treated with DSS alone or ApcMin mice treated with DSS.
Mentions: In order to determine whether aberrant β-catenin expression/localization correlated with MUC4 expression in CRC, we first examined the expression of MUC4 and β-catenin in CRC cell lines. While all cell lines examined expressed β-catenin, only two of the 7 cell lines were found to express MUC4 abundantly; LS180 and HCT-8 (Figure 1A), while MUC4 expression in HCT116 was negligible to none. However, with confocal microscopy, HCT116 was observed to express low levels of MUC4, in contrast with the HCT-8 cell line, where MUC4 was abundantly expressed (Figure 1B). Thus, a majority of the CRC cell lines examined displayed a loss of MUC4.

View Article: PubMed Central - PubMed

ABSTRACT

MUC4 is a transmembrane mucin lining the normal colonic epithelium. The aberrant/de novo over-expression of MUC4 is well documented in malignancies of the pancreas, ovary and breast. However, studies have reported the loss of MUC4 expression in the majority of colorectal cancers (CRCs). A MUC4 promoter analysis showed the presence of three putative TCF/LEF sites, implying a possible regulation by the Wnt/β-catenin pathway, which has been shown to drive CRC progression. Thus, the objective of our study was to determine whether MUC4 is regulated by β-catenin in CRC. We first knocked down (KD) β-catenin in three CRC cell lines; LS180, HCT-8 and HCT116, which resulted in increased MUC4 transcript and MUC4 protein. Additionally, the overexpression of stabilized mutant β-catenin in LS180 and HCT-8 resulted in a decrease in MUC4 expression. Immunohistochemistry (IHC) of mouse colon tissue harboring tubular adenomas and high grade dysplasia showed dramatically reduced Muc4 in lesions relative to adjacent normal tissue, with increased cytosolic/nuclear β-catenin. Luciferase assays with the complete MUC4 promoter construct p3778 showed increased MUC4 promoter luciferase activity in the absence of β-catenin (KD). Mutation of all three putative TCF/LEF sites showed that MUC4 promoter luciferase activity was increased relative to the un-mutated promoter. Interestingly, it was observed that MUC4 expressing CRC cell lines also expressed high levels of Hath1, a transcription factor repressed by both active Wnt/β-catenin and Notch signaling. The KD of β-catenin and/or treatment with a Notch γ-secretase inhibitor, Dibenzazepine (DBZ) resulted in increased Hath1 and MUC4 in LS180, HCT-8 and HCT116. Furthermore, overexpression of Hath1 in HCT-8 and LS180 caused increased MUC4 transcript and MUC4 protein. Taken together, our results indicate that the Wnt/β-catenin pathway suppresses the Notch pathway effector Hath1, resulting in reduced MUC4 in CRC.

No MeSH data available.