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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

Addition of NLS inactivates the potential of DNCT.(a) Schematic representation of DNCTNLS, a derivative of DNCT with the SV40 large T antigen at the C-terminus. DNCTNLS and DNCT both have a FLAG tag at their C-termini. (b) Immunoblot analysis revealed that comparable amounts of DNCT and DNCTNLS were produced by the stable transfectants. The blots were probed with anti-vinculin (a loading control) and anti-FLAG antibodies. (c) Immunoblot analysis revealed that the levels of E-cadherin, α-catenin, and β-catenin do not change upon DNCTNLS expression. (d) DNCTNLS does not impair the cell surface transport of endogenous E-cadherin. E-cadherin was detected with DECMA-1. (e) DNCTNLS induces the nuclear accumulation of β-catenin, but does not deplete β-catenin from the cell surface. (f) As in the case of β-catenin, a significant portion of α-catenin colocalized with DNCTNLS in the nucleus, but Merlin remained on the cell-surface membrane. Thus, the interaction of Merlin with α-catenin takes place in the cytoplasm, but not in the nucleus. Bars, 25 μm. (g) DNCTNLS does not inhibit anoikis. Cells were cultured in suspension in the presence (+) or absence (−) of Dox for 3 d, and then stained with FITC-annexin V. (h) DNCTNLS failed to enhance ERK signaling. DNCTNLS+ cells were cultured on normal plates (attached) or on ultra-low attachment plates (suspension) in the presence (+) or absence (−) of Dox. Then, the cells were harvested, and extracts were analyzed by immunoblot using the indicated antibodies. In (c,h), vinculin was used as a loading control.
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f7: Addition of NLS inactivates the potential of DNCT.(a) Schematic representation of DNCTNLS, a derivative of DNCT with the SV40 large T antigen at the C-terminus. DNCTNLS and DNCT both have a FLAG tag at their C-termini. (b) Immunoblot analysis revealed that comparable amounts of DNCT and DNCTNLS were produced by the stable transfectants. The blots were probed with anti-vinculin (a loading control) and anti-FLAG antibodies. (c) Immunoblot analysis revealed that the levels of E-cadherin, α-catenin, and β-catenin do not change upon DNCTNLS expression. (d) DNCTNLS does not impair the cell surface transport of endogenous E-cadherin. E-cadherin was detected with DECMA-1. (e) DNCTNLS induces the nuclear accumulation of β-catenin, but does not deplete β-catenin from the cell surface. (f) As in the case of β-catenin, a significant portion of α-catenin colocalized with DNCTNLS in the nucleus, but Merlin remained on the cell-surface membrane. Thus, the interaction of Merlin with α-catenin takes place in the cytoplasm, but not in the nucleus. Bars, 25 μm. (g) DNCTNLS does not inhibit anoikis. Cells were cultured in suspension in the presence (+) or absence (−) of Dox for 3 d, and then stained with FITC-annexin V. (h) DNCTNLS failed to enhance ERK signaling. DNCTNLS+ cells were cultured on normal plates (attached) or on ultra-low attachment plates (suspension) in the presence (+) or absence (−) of Dox. Then, the cells were harvested, and extracts were analyzed by immunoblot using the indicated antibodies. In (c,h), vinculin was used as a loading control.

Mentions: It has been shown that the PS1-dependent ε-cleavage product of N-cadherin, the soluble N-cadherin cytoplasmic domain, binds the transcription factor CBP (CREB-binding protein), promotes its proteasomal degradation, and inhibits CRE-dependent transactivation43. Thus, the domain functions as a potent repressor of CBP/CREB (cyclic AMP responsive element–binding protein)-mediated transcription. Intriguingly and in contrast to those reports, the soluble cytoplasmic N-cadherin domain has also been shown to translocate to the nucleus and enhance β-catenin signaling4445. Furthermore, nuclear expression of the E-cadherin cytoplasmic domain tagged with NLS has been shown to suppress staurosporine-induced apoptosis46. Therefore, we asked whether nuclear localization of DNCT facilitated the inhibition of anoikis. To this end, we made a construct encoding DNCT with the SV40 large T antigen NLS at its C-terminus (DNCTNLS; Fig. 7a), and introduced it into T23 MDCK cells. Although immunoblot analysis revealed that comparable amounts of DNCT and DNCTNLS were produced by the stable transfectants (Fig. 7b), DNCTNLS expression did not change the levels of E-cadherin, α-catenin, or β-catenin (Fig. 7c), nor did not impair the cell-surface transport of endogenous E-cadherin (Fig. 7d), or deplete β-catenin from the cell surface (Fig. 7e). As in the case of β-catenin, a significant fraction of α-catenin colocalized with DNCTNLS in the nucleus, whereas Merlin remained on the cell surface (Fig. 7f). More importantly, after 3 d in suspension culture, a majority (>80%) of DNCTNLS+ cells with or without Dox treatment were stained by annexin V (Fig. 7g). Thus, the addition of NLS to DNCT inactivated its ability to suppress anoikis. Consistent with these observations, DNCTNLS failed to enhance ERK signaling under both normal (attached) and suspension conditions (Fig. 7h).


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

Ozawa M - Sci Rep (2015)

Addition of NLS inactivates the potential of DNCT.(a) Schematic representation of DNCTNLS, a derivative of DNCT with the SV40 large T antigen at the C-terminus. DNCTNLS and DNCT both have a FLAG tag at their C-termini. (b) Immunoblot analysis revealed that comparable amounts of DNCT and DNCTNLS were produced by the stable transfectants. The blots were probed with anti-vinculin (a loading control) and anti-FLAG antibodies. (c) Immunoblot analysis revealed that the levels of E-cadherin, α-catenin, and β-catenin do not change upon DNCTNLS expression. (d) DNCTNLS does not impair the cell surface transport of endogenous E-cadherin. E-cadherin was detected with DECMA-1. (e) DNCTNLS induces the nuclear accumulation of β-catenin, but does not deplete β-catenin from the cell surface. (f) As in the case of β-catenin, a significant portion of α-catenin colocalized with DNCTNLS in the nucleus, but Merlin remained on the cell-surface membrane. Thus, the interaction of Merlin with α-catenin takes place in the cytoplasm, but not in the nucleus. Bars, 25 μm. (g) DNCTNLS does not inhibit anoikis. Cells were cultured in suspension in the presence (+) or absence (−) of Dox for 3 d, and then stained with FITC-annexin V. (h) DNCTNLS failed to enhance ERK signaling. DNCTNLS+ cells were cultured on normal plates (attached) or on ultra-low attachment plates (suspension) in the presence (+) or absence (−) of Dox. Then, the cells were harvested, and extracts were analyzed by immunoblot using the indicated antibodies. In (c,h), vinculin was used as a loading control.
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f7: Addition of NLS inactivates the potential of DNCT.(a) Schematic representation of DNCTNLS, a derivative of DNCT with the SV40 large T antigen at the C-terminus. DNCTNLS and DNCT both have a FLAG tag at their C-termini. (b) Immunoblot analysis revealed that comparable amounts of DNCT and DNCTNLS were produced by the stable transfectants. The blots were probed with anti-vinculin (a loading control) and anti-FLAG antibodies. (c) Immunoblot analysis revealed that the levels of E-cadherin, α-catenin, and β-catenin do not change upon DNCTNLS expression. (d) DNCTNLS does not impair the cell surface transport of endogenous E-cadherin. E-cadherin was detected with DECMA-1. (e) DNCTNLS induces the nuclear accumulation of β-catenin, but does not deplete β-catenin from the cell surface. (f) As in the case of β-catenin, a significant portion of α-catenin colocalized with DNCTNLS in the nucleus, but Merlin remained on the cell-surface membrane. Thus, the interaction of Merlin with α-catenin takes place in the cytoplasm, but not in the nucleus. Bars, 25 μm. (g) DNCTNLS does not inhibit anoikis. Cells were cultured in suspension in the presence (+) or absence (−) of Dox for 3 d, and then stained with FITC-annexin V. (h) DNCTNLS failed to enhance ERK signaling. DNCTNLS+ cells were cultured on normal plates (attached) or on ultra-low attachment plates (suspension) in the presence (+) or absence (−) of Dox. Then, the cells were harvested, and extracts were analyzed by immunoblot using the indicated antibodies. In (c,h), vinculin was used as a loading control.
Mentions: It has been shown that the PS1-dependent ε-cleavage product of N-cadherin, the soluble N-cadherin cytoplasmic domain, binds the transcription factor CBP (CREB-binding protein), promotes its proteasomal degradation, and inhibits CRE-dependent transactivation43. Thus, the domain functions as a potent repressor of CBP/CREB (cyclic AMP responsive element–binding protein)-mediated transcription. Intriguingly and in contrast to those reports, the soluble cytoplasmic N-cadherin domain has also been shown to translocate to the nucleus and enhance β-catenin signaling4445. Furthermore, nuclear expression of the E-cadherin cytoplasmic domain tagged with NLS has been shown to suppress staurosporine-induced apoptosis46. Therefore, we asked whether nuclear localization of DNCT facilitated the inhibition of anoikis. To this end, we made a construct encoding DNCT with the SV40 large T antigen NLS at its C-terminus (DNCTNLS; Fig. 7a), and introduced it into T23 MDCK cells. Although immunoblot analysis revealed that comparable amounts of DNCT and DNCTNLS were produced by the stable transfectants (Fig. 7b), DNCTNLS expression did not change the levels of E-cadherin, α-catenin, or β-catenin (Fig. 7c), nor did not impair the cell-surface transport of endogenous E-cadherin (Fig. 7d), or deplete β-catenin from the cell surface (Fig. 7e). As in the case of β-catenin, a significant fraction of α-catenin colocalized with DNCTNLS in the nucleus, whereas Merlin remained on the cell surface (Fig. 7f). More importantly, after 3 d in suspension culture, a majority (>80%) of DNCTNLS+ cells with or without Dox treatment were stained by annexin V (Fig. 7g). Thus, the addition of NLS to DNCT inactivated its ability to suppress anoikis. Consistent with these observations, DNCTNLS failed to enhance ERK signaling under both normal (attached) and suspension conditions (Fig. 7h).

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