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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.


Related in: MedlinePlus

A schematic representation of the findings of this study(A) In the normal colon, Wnt signaling is inactive and Hath1 is transcriptionally active in differentiated secretory cells. Hath1 transcriptionally activates MUC4, as well as other mucins such as MUC2. (B) However, in colorectal cancer, active Wnt/β-catenin and Notch signaling transcriptionally upregulate Hes1. Hes1 antagonizes Hath1, thus reducing Hath1 levels. In addition, the Hath1 protein is targeted for phosphorylation mediated destruction by GSK3β instead of β-catenin, further reducing Hath1 levels. Thus, MUC4 is no longer actively transcribed owing to reduced Hath1, resulting in reduced MUC4.
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Figure 7: A schematic representation of the findings of this study(A) In the normal colon, Wnt signaling is inactive and Hath1 is transcriptionally active in differentiated secretory cells. Hath1 transcriptionally activates MUC4, as well as other mucins such as MUC2. (B) However, in colorectal cancer, active Wnt/β-catenin and Notch signaling transcriptionally upregulate Hes1. Hes1 antagonizes Hath1, thus reducing Hath1 levels. In addition, the Hath1 protein is targeted for phosphorylation mediated destruction by GSK3β instead of β-catenin, further reducing Hath1 levels. Thus, MUC4 is no longer actively transcribed owing to reduced Hath1, resulting in reduced MUC4.

Mentions: In summation, our results suggest that Hath1 may regulate MUC4 and that during the course of CRC progression, both the Notch and Wnt pathways converge to repress Hath1 [15, 18]. This results in decreased MUC4 transcription. The conclusions from this study have been summarized in Figure 7. In the normal differentiated secretory colonocyte (Figure 7A), Hath1 activates MUC4 while the Wnt/β-catenin pathway is inactive. In CRC (Figure 7B), β-catenin enters the nucleus and up-regulates Hes1 [15], while the Notch pathway also activates Hes1, which antagonizes Hath1 [26, 45]. Meanwhile, Hath1 is also targeted for destruction by GSK3β mediated phosphorylation [18]. Thus, Hath1 mediated MUC4 up- regulation is abrogated, resulting in reduced MUC4. In conclusion, this study shows for the first time that Wnt/β- catenin can repress MUC4 in CRC via the repression of the Notch effector Hath1, which ordinarily governs MUC4 in the normal colonocyte.


MUC4 is negatively regulated through the Wnt/ β -catenin pathway via the Notch effector Hath1 in colorectal cancer
A schematic representation of the findings of this study(A) In the normal colon, Wnt signaling is inactive and Hath1 is transcriptionally active in differentiated secretory cells. Hath1 transcriptionally activates MUC4, as well as other mucins such as MUC2. (B) However, in colorectal cancer, active Wnt/β-catenin and Notch signaling transcriptionally upregulate Hes1. Hes1 antagonizes Hath1, thus reducing Hath1 levels. In addition, the Hath1 protein is targeted for phosphorylation mediated destruction by GSK3β instead of β-catenin, further reducing Hath1 levels. Thus, MUC4 is no longer actively transcribed owing to reduced Hath1, resulting in reduced MUC4.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: A schematic representation of the findings of this study(A) In the normal colon, Wnt signaling is inactive and Hath1 is transcriptionally active in differentiated secretory cells. Hath1 transcriptionally activates MUC4, as well as other mucins such as MUC2. (B) However, in colorectal cancer, active Wnt/β-catenin and Notch signaling transcriptionally upregulate Hes1. Hes1 antagonizes Hath1, thus reducing Hath1 levels. In addition, the Hath1 protein is targeted for phosphorylation mediated destruction by GSK3β instead of β-catenin, further reducing Hath1 levels. Thus, MUC4 is no longer actively transcribed owing to reduced Hath1, resulting in reduced MUC4.
Mentions: In summation, our results suggest that Hath1 may regulate MUC4 and that during the course of CRC progression, both the Notch and Wnt pathways converge to repress Hath1 [15, 18]. This results in decreased MUC4 transcription. The conclusions from this study have been summarized in Figure 7. In the normal differentiated secretory colonocyte (Figure 7A), Hath1 activates MUC4 while the Wnt/β-catenin pathway is inactive. In CRC (Figure 7B), β-catenin enters the nucleus and up-regulates Hes1 [15], while the Notch pathway also activates Hes1, which antagonizes Hath1 [26, 45]. Meanwhile, Hath1 is also targeted for destruction by GSK3β mediated phosphorylation [18]. Thus, Hath1 mediated MUC4 up- regulation is abrogated, resulting in reduced MUC4. In conclusion, this study shows for the first time that Wnt/β- catenin can repress MUC4 in CRC via the repression of the Notch effector Hath1, which ordinarily governs MUC4 in the normal colonocyte.

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.


Related in: MedlinePlus