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The N-cadherin cytoplasmic domain confers anchorage-independent growth and the loss of contact inhibition.

Ozawa M - Sci Rep (2015)

Bottom Line: Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated.An E-cadherin-α-catenin chimera that functions as a β-catenin-independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth.Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan.

ABSTRACT
Tumor growth is characterized by anchorage independence and the loss of contact inhibition. Previously, we showed that either a red fluorescent protein (DsRed)-tagged N-cadherin or E-cadherin cytoplasmic domain (DNCT or DECT) could function as a dominant negative inhibitor by blocking the cell surface localization of endogenous E-cadherin and inducing cell dissociation. Here, we show that expression of DNCT abrogated contact inhibition of proliferation and conferred anchorage-independent growth. DNCT expression induced the relocation of the tumor suppressor Merlin from the cell surface to intracellular compartments. Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated. An E-cadherin-α-catenin chimera that functions as a β-catenin-independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth. Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus.

No MeSH data available.


Related in: MedlinePlus

MAP kinase is involved in DNCT-induced protection from anoikis.(a) A MEK inhibitor impairs DNCT-induced survival in suspension cultures. A proliferation assay was carried out in the presence of the following chemicals: DMSO (control), PD0325901 (1 μM), Wortmannin (10 μM), LY294002 (25 μM), AKT inhibitor V (30 μM), AKT 1/2 (30 μM), and staurosporine (an apoptosis inducer; 1 μM). DNCT+ cells were cultured on ultra-low attachment plates in the presence (+) or absence (−) of Dox. After 72 h, cells were assayed for their viability using the WST-1 assay. The results were expressed as a percentage of control (DMSO) cells cultured in the absence of Dox (−Dox). Values represent the mean ± S.E.; n = 3. (b) The levels of activated MAP kinase and cyclin D1 remained elevated in DNCT+ (−Dox) cells. Cells were cultured on ultra-low attachment plates (in suspension) in the presence (+) or absence (−) of Dox for 24 h. Then, the cells were harvested, and extracts were analyzed by immunoblot using the indicated antibodies. (c) The MEK inhibitor PD0325901 blocks phosphorylation of ERK. Cells were cultured on ultra-low attachment plates (suspension) or normal plates (attached) in the presence (+) or absence (−) of PD0325901 (1 μM) for 24 h. After the cells were harvested, extracts were analyzed by immunoblotting using the indicated antibodies. In (b,c), vinculin was used as a loading control.
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f3: MAP kinase is involved in DNCT-induced protection from anoikis.(a) A MEK inhibitor impairs DNCT-induced survival in suspension cultures. A proliferation assay was carried out in the presence of the following chemicals: DMSO (control), PD0325901 (1 μM), Wortmannin (10 μM), LY294002 (25 μM), AKT inhibitor V (30 μM), AKT 1/2 (30 μM), and staurosporine (an apoptosis inducer; 1 μM). DNCT+ cells were cultured on ultra-low attachment plates in the presence (+) or absence (−) of Dox. After 72 h, cells were assayed for their viability using the WST-1 assay. The results were expressed as a percentage of control (DMSO) cells cultured in the absence of Dox (−Dox). Values represent the mean ± S.E.; n = 3. (b) The levels of activated MAP kinase and cyclin D1 remained elevated in DNCT+ (−Dox) cells. Cells were cultured on ultra-low attachment plates (in suspension) in the presence (+) or absence (−) of Dox for 24 h. Then, the cells were harvested, and extracts were analyzed by immunoblot using the indicated antibodies. (c) The MEK inhibitor PD0325901 blocks phosphorylation of ERK. Cells were cultured on ultra-low attachment plates (suspension) or normal plates (attached) in the presence (+) or absence (−) of PD0325901 (1 μM) for 24 h. After the cells were harvested, extracts were analyzed by immunoblotting using the indicated antibodies. In (b,c), vinculin was used as a loading control.

Mentions: DNCT expression led to the activation of MAP kinase and Akt signaling; these signaling pathways have been implicated in the promotion of cell proliferation and survival29. Therefore, we tested whether DNCT-mediated protection against anoikis was dependent on these signaling pathways. Indeed, treatment with PD0325901, a potent inhibitor of MAP kinase kinase (MEK), significantly abolished the protective effect of DNCT expression on cells cultured in suspension (Fig. 3a), suggesting that MAP kinase signaling is involved in the rescue of cells from anoikis by DNCT. Treatment with the PI3 kinase inhibitors, Wortmannin and LY294002, slightly inhibited cell growth in the same assay, while treatment with other inhibitors of cell proliferation–related signaling pathways did not significantly influence viability.


The N-cadherin cytoplasmic domain confers anchorage-independent growth and the loss of contact inhibition.

Ozawa M - Sci Rep (2015)

MAP kinase is involved in DNCT-induced protection from anoikis.(a) A MEK inhibitor impairs DNCT-induced survival in suspension cultures. A proliferation assay was carried out in the presence of the following chemicals: DMSO (control), PD0325901 (1 μM), Wortmannin (10 μM), LY294002 (25 μM), AKT inhibitor V (30 μM), AKT 1/2 (30 μM), and staurosporine (an apoptosis inducer; 1 μM). DNCT+ cells were cultured on ultra-low attachment plates in the presence (+) or absence (−) of Dox. After 72 h, cells were assayed for their viability using the WST-1 assay. The results were expressed as a percentage of control (DMSO) cells cultured in the absence of Dox (−Dox). Values represent the mean ± S.E.; n = 3. (b) The levels of activated MAP kinase and cyclin D1 remained elevated in DNCT+ (−Dox) cells. Cells were cultured on ultra-low attachment plates (in suspension) in the presence (+) or absence (−) of Dox for 24 h. Then, the cells were harvested, and extracts were analyzed by immunoblot using the indicated antibodies. (c) The MEK inhibitor PD0325901 blocks phosphorylation of ERK. Cells were cultured on ultra-low attachment plates (suspension) or normal plates (attached) in the presence (+) or absence (−) of PD0325901 (1 μM) for 24 h. After the cells were harvested, extracts were analyzed by immunoblotting using the indicated antibodies. In (b,c), vinculin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4612716&req=5

f3: MAP kinase is involved in DNCT-induced protection from anoikis.(a) A MEK inhibitor impairs DNCT-induced survival in suspension cultures. A proliferation assay was carried out in the presence of the following chemicals: DMSO (control), PD0325901 (1 μM), Wortmannin (10 μM), LY294002 (25 μM), AKT inhibitor V (30 μM), AKT 1/2 (30 μM), and staurosporine (an apoptosis inducer; 1 μM). DNCT+ cells were cultured on ultra-low attachment plates in the presence (+) or absence (−) of Dox. After 72 h, cells were assayed for their viability using the WST-1 assay. The results were expressed as a percentage of control (DMSO) cells cultured in the absence of Dox (−Dox). Values represent the mean ± S.E.; n = 3. (b) The levels of activated MAP kinase and cyclin D1 remained elevated in DNCT+ (−Dox) cells. Cells were cultured on ultra-low attachment plates (in suspension) in the presence (+) or absence (−) of Dox for 24 h. Then, the cells were harvested, and extracts were analyzed by immunoblot using the indicated antibodies. (c) The MEK inhibitor PD0325901 blocks phosphorylation of ERK. Cells were cultured on ultra-low attachment plates (suspension) or normal plates (attached) in the presence (+) or absence (−) of PD0325901 (1 μM) for 24 h. After the cells were harvested, extracts were analyzed by immunoblotting using the indicated antibodies. In (b,c), vinculin was used as a loading control.
Mentions: DNCT expression led to the activation of MAP kinase and Akt signaling; these signaling pathways have been implicated in the promotion of cell proliferation and survival29. Therefore, we tested whether DNCT-mediated protection against anoikis was dependent on these signaling pathways. Indeed, treatment with PD0325901, a potent inhibitor of MAP kinase kinase (MEK), significantly abolished the protective effect of DNCT expression on cells cultured in suspension (Fig. 3a), suggesting that MAP kinase signaling is involved in the rescue of cells from anoikis by DNCT. Treatment with the PI3 kinase inhibitors, Wortmannin and LY294002, slightly inhibited cell growth in the same assay, while treatment with other inhibitors of cell proliferation–related signaling pathways did not significantly influence viability.

Bottom Line: Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated.An E-cadherin-α-catenin chimera that functions as a β-catenin-independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth.Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan.

ABSTRACT
Tumor growth is characterized by anchorage independence and the loss of contact inhibition. Previously, we showed that either a red fluorescent protein (DsRed)-tagged N-cadherin or E-cadherin cytoplasmic domain (DNCT or DECT) could function as a dominant negative inhibitor by blocking the cell surface localization of endogenous E-cadherin and inducing cell dissociation. Here, we show that expression of DNCT abrogated contact inhibition of proliferation and conferred anchorage-independent growth. DNCT expression induced the relocation of the tumor suppressor Merlin from the cell surface to intracellular compartments. Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated. An E-cadherin-α-catenin chimera that functions as a β-catenin-independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth. Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus.

No MeSH data available.


Related in: MedlinePlus