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Mucin1 shifts Smad3 signaling from the tumor-suppressive pSmad3C/p21(WAF1) pathway to the oncogenic pSmad3L/c-Myc pathway by activating JNK in human hepatocellular carcinoma cells.

Li Q, Liu G, Yuan H, Wang J, Guo Y, Chen T, Zhai R, Shao D, Ni W, Tai G - Oncotarget (2015)

Bottom Line: Conversely, MUC1 gene silencing in MUC1 expressing HCC cells results in preserved tumor-suppressive function via pSmad3C, while eliminating pSmad3L-mediated oncogenic activity both in vitro and in vivo.In addition, high correlation between MUC1 and pSmad3L/c-Myc but not pSmad3C/p21(WAF1) expression was observed in HCC tissues from patients.Collectively, these results indicate that MUC1 shifts Smad3 signaling from a tumor-suppressive pSmad3C/p21(WAF1) to an oncogenic pSmad3L/c-Myc pathway by directly activating JNK in HCC cells, suggesting that MUC1 is an important target for HCC therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, College of Basic Medical Science, Jilin University, Changchun 130021, China.

ABSTRACT
Mucin1 (MUC1) is a transmembrane glycoprotein that acts as an oncogene in human hepatic tumorigenesis. Hepatocellular carcinoma (HCC) cells often gain advantage by reducing the tumor-suppressive activity of transforming growth factor beta (TGF-β) together with stimulation of its oncogenic activity as in MUC1 expressing HCC cells; however, molecular mechanisms remain largely unknown. Type I TGF-β receptor (TβRI) and c-Jun NH2-terminal kinase (JNK) differentially phosphorylate Smad3 mediator to create 2 phosphorylated forms: COOH-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). Here, we report that MUC1 overexpression in HCC cell lines suppresses TβRI-mediated pSmad3C signaling which involves growth inhibition by up-regulating p21(WAF1). Instead, MUC1 directly activates JNK to stimulate oncogenic pSmad3L signaling, which fosters cell proliferation by up-regulating c-Myc. Conversely, MUC1 gene silencing in MUC1 expressing HCC cells results in preserved tumor-suppressive function via pSmad3C, while eliminating pSmad3L-mediated oncogenic activity both in vitro and in vivo. In addition, high correlation between MUC1 and pSmad3L/c-Myc but not pSmad3C/p21(WAF1) expression was observed in HCC tissues from patients. Collectively, these results indicate that MUC1 shifts Smad3 signaling from a tumor-suppressive pSmad3C/p21(WAF1) to an oncogenic pSmad3L/c-Myc pathway by directly activating JNK in HCC cells, suggesting that MUC1 is an important target for HCC therapy.

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TGF-β has no effect on the MUC1-induced switch in Smad3 signaling in HCC cells(A) Bel7402, 7402-EV and 7402-MUC1 cells were serum-starved for 16 h before treatment with exogenous TGF-β1 (50 pM) and the TβR inhibitor SB431542 (20 μM) for 48 h, and cell viability was determined using the WST-1 assay. The data are expressed as the means ± SD of three independent experiments. **P < 0.01 compared with the 7402-MUC1 cells. (B, C and D) The 7402-EV and 7402-MUC1 cells were serum-starved for 16 h before treatment with or without exogenous TGF-β1 (50 pM) and TβR inhibitor SB431542 (20 μM), and analysis by Western blotting for the expression of p-JNK (B), p-Smad3L (Ser-213) and p-Smad3C (Ser-423/425) (C and D). GAPDH was used as the loading control. The results are representative of three independent experiments.
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Figure 4: TGF-β has no effect on the MUC1-induced switch in Smad3 signaling in HCC cells(A) Bel7402, 7402-EV and 7402-MUC1 cells were serum-starved for 16 h before treatment with exogenous TGF-β1 (50 pM) and the TβR inhibitor SB431542 (20 μM) for 48 h, and cell viability was determined using the WST-1 assay. The data are expressed as the means ± SD of three independent experiments. **P < 0.01 compared with the 7402-MUC1 cells. (B, C and D) The 7402-EV and 7402-MUC1 cells were serum-starved for 16 h before treatment with or without exogenous TGF-β1 (50 pM) and TβR inhibitor SB431542 (20 μM), and analysis by Western blotting for the expression of p-JNK (B), p-Smad3L (Ser-213) and p-Smad3C (Ser-423/425) (C and D). GAPDH was used as the loading control. The results are representative of three independent experiments.

Mentions: Studies have shown that TGF-β can activate JNK by mediating Smad-independent signaling [33, 34], and our previous study showed that MUC1 expression induces TGF-β1 secretion (data not shown), leading to a hypothesis that MUC1-induced TGF-β secretion influences JNK activation and then mediates Smad3 signaling. Thus, the 7402-EV and 7402-MUC1 cells were treated with exogenous TGF-β1 and TGF-β receptor (TβR) inhibitor (SB431542). Then, the cell viability was analyzed by WST-1, and the Smad3 signaling molecules and JNK activation were detected by Western blotting. The effects of exogenous TGF-β1 and TβR inhibitor on cell proliferation and JNK activation were not observed in the MUC1-overexpressing cells (Figure 4A and 4B). Moreover, TGF-β1 enhanced the levels of both pSmad3L (Ser-213) and pSmad3C (Ser-423/425), which were inhibited by the TβR inhibitor in both 7402-EV and 7402-MUC1 cells (Figure 4C and 4D). These results indicate that JNK activation is dependent on MUC1 but not TGF-β, and TGF-β has no effect on MUC1-induced switch in Smad3 signaling in HCC cells.


Mucin1 shifts Smad3 signaling from the tumor-suppressive pSmad3C/p21(WAF1) pathway to the oncogenic pSmad3L/c-Myc pathway by activating JNK in human hepatocellular carcinoma cells.

Li Q, Liu G, Yuan H, Wang J, Guo Y, Chen T, Zhai R, Shao D, Ni W, Tai G - Oncotarget (2015)

TGF-β has no effect on the MUC1-induced switch in Smad3 signaling in HCC cells(A) Bel7402, 7402-EV and 7402-MUC1 cells were serum-starved for 16 h before treatment with exogenous TGF-β1 (50 pM) and the TβR inhibitor SB431542 (20 μM) for 48 h, and cell viability was determined using the WST-1 assay. The data are expressed as the means ± SD of three independent experiments. **P < 0.01 compared with the 7402-MUC1 cells. (B, C and D) The 7402-EV and 7402-MUC1 cells were serum-starved for 16 h before treatment with or without exogenous TGF-β1 (50 pM) and TβR inhibitor SB431542 (20 μM), and analysis by Western blotting for the expression of p-JNK (B), p-Smad3L (Ser-213) and p-Smad3C (Ser-423/425) (C and D). GAPDH was used as the loading control. The results are representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 4: TGF-β has no effect on the MUC1-induced switch in Smad3 signaling in HCC cells(A) Bel7402, 7402-EV and 7402-MUC1 cells were serum-starved for 16 h before treatment with exogenous TGF-β1 (50 pM) and the TβR inhibitor SB431542 (20 μM) for 48 h, and cell viability was determined using the WST-1 assay. The data are expressed as the means ± SD of three independent experiments. **P < 0.01 compared with the 7402-MUC1 cells. (B, C and D) The 7402-EV and 7402-MUC1 cells were serum-starved for 16 h before treatment with or without exogenous TGF-β1 (50 pM) and TβR inhibitor SB431542 (20 μM), and analysis by Western blotting for the expression of p-JNK (B), p-Smad3L (Ser-213) and p-Smad3C (Ser-423/425) (C and D). GAPDH was used as the loading control. The results are representative of three independent experiments.
Mentions: Studies have shown that TGF-β can activate JNK by mediating Smad-independent signaling [33, 34], and our previous study showed that MUC1 expression induces TGF-β1 secretion (data not shown), leading to a hypothesis that MUC1-induced TGF-β secretion influences JNK activation and then mediates Smad3 signaling. Thus, the 7402-EV and 7402-MUC1 cells were treated with exogenous TGF-β1 and TGF-β receptor (TβR) inhibitor (SB431542). Then, the cell viability was analyzed by WST-1, and the Smad3 signaling molecules and JNK activation were detected by Western blotting. The effects of exogenous TGF-β1 and TβR inhibitor on cell proliferation and JNK activation were not observed in the MUC1-overexpressing cells (Figure 4A and 4B). Moreover, TGF-β1 enhanced the levels of both pSmad3L (Ser-213) and pSmad3C (Ser-423/425), which were inhibited by the TβR inhibitor in both 7402-EV and 7402-MUC1 cells (Figure 4C and 4D). These results indicate that JNK activation is dependent on MUC1 but not TGF-β, and TGF-β has no effect on MUC1-induced switch in Smad3 signaling in HCC cells.

Bottom Line: Conversely, MUC1 gene silencing in MUC1 expressing HCC cells results in preserved tumor-suppressive function via pSmad3C, while eliminating pSmad3L-mediated oncogenic activity both in vitro and in vivo.In addition, high correlation between MUC1 and pSmad3L/c-Myc but not pSmad3C/p21(WAF1) expression was observed in HCC tissues from patients.Collectively, these results indicate that MUC1 shifts Smad3 signaling from a tumor-suppressive pSmad3C/p21(WAF1) to an oncogenic pSmad3L/c-Myc pathway by directly activating JNK in HCC cells, suggesting that MUC1 is an important target for HCC therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, College of Basic Medical Science, Jilin University, Changchun 130021, China.

ABSTRACT
Mucin1 (MUC1) is a transmembrane glycoprotein that acts as an oncogene in human hepatic tumorigenesis. Hepatocellular carcinoma (HCC) cells often gain advantage by reducing the tumor-suppressive activity of transforming growth factor beta (TGF-β) together with stimulation of its oncogenic activity as in MUC1 expressing HCC cells; however, molecular mechanisms remain largely unknown. Type I TGF-β receptor (TβRI) and c-Jun NH2-terminal kinase (JNK) differentially phosphorylate Smad3 mediator to create 2 phosphorylated forms: COOH-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). Here, we report that MUC1 overexpression in HCC cell lines suppresses TβRI-mediated pSmad3C signaling which involves growth inhibition by up-regulating p21(WAF1). Instead, MUC1 directly activates JNK to stimulate oncogenic pSmad3L signaling, which fosters cell proliferation by up-regulating c-Myc. Conversely, MUC1 gene silencing in MUC1 expressing HCC cells results in preserved tumor-suppressive function via pSmad3C, while eliminating pSmad3L-mediated oncogenic activity both in vitro and in vivo. In addition, high correlation between MUC1 and pSmad3L/c-Myc but not pSmad3C/p21(WAF1) expression was observed in HCC tissues from patients. Collectively, these results indicate that MUC1 shifts Smad3 signaling from a tumor-suppressive pSmad3C/p21(WAF1) to an oncogenic pSmad3L/c-Myc pathway by directly activating JNK in HCC cells, suggesting that MUC1 is an important target for HCC therapy.

Show MeSH
Related in: MedlinePlus