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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

DNCT expression inhibits YAP/TAZ and β-catenin transcriptional activity.A YAP/TAZ-responsive EGFP reporter (8 × GTIIC-EGFP) (a) or a β-catenin–responsive EGFP reporter (TOP-EGFP) (b) was introduced into DNCT+ cells and stable transfectants were isolated. Cells were cultured for 2 d in the presence (+) or absence (−) of Dox prior to the initiation of the experiments. Then, cells were cultured for 24 h in the presence (+) or absence (−) of LPA (20 μM) or BIO (3 μM) to stimulate YAP/TAZ or β-catenin transcriptional activity, respectively, and were analyzed by flow cytometry.
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f6: DNCT expression inhibits YAP/TAZ and β-catenin transcriptional activity.A YAP/TAZ-responsive EGFP reporter (8 × GTIIC-EGFP) (a) or a β-catenin–responsive EGFP reporter (TOP-EGFP) (b) was introduced into DNCT+ cells and stable transfectants were isolated. Cells were cultured for 2 d in the presence (+) or absence (−) of Dox prior to the initiation of the experiments. Then, cells were cultured for 24 h in the presence (+) or absence (−) of LPA (20 μM) or BIO (3 μM) to stimulate YAP/TAZ or β-catenin transcriptional activity, respectively, and were analyzed by flow cytometry.

Mentions: To determine whether TAZ activity is regulated by DNCT expression, we monitored TAZ transcriptional activity in DNCT+ cells cultured with or without Dox. For this, we used a synthetic YAP/TAZ-responsive EGFP reporter (8×GTIIC-EGFP) as a direct read-out of activity. A similar construct was previously used to successfully monitor TEAD-dependent YAP/TAZ activity24. The present construct was introduced into DNCT+ cells and stable transfectants were isolated. 8×GTIIC-EGFP/DNCT cells cultured in the presence of Dox and without serum exhibited low levels of EGFP protein expression as determined by flow cytometry (Fig. 6a, +Dox/-serum). Lysophosphatidic acid (LPA) has recently shown to activate the YAP/TAZ transcription activity37, and the addition of LPA to cultures increased expression of the EGFP protein (Fig. 6a, +Dox/+LPA). When the cells were cultured in the absence of Dox, EGFP levels were decreased (Fig. 6a, -Dox/-serum), and YAP/TAZ activity was not activated by the addition of LPA (Fig. 6a, -Dox/+LPA). Consistent with these results, a comparison of the gene expression of DNCT+ cells cultured with or without Dox using an Agilent Whole Canine microarray revealed no change in the expression of CTGF (Table S1). Of the 112 transcripts previously identified as significantly increased by YAP overexpression in NIH 3T3 cells19, four were upregulated and three downregulated by DNCT expression; the rest (105 genes) exhibited no change (Table S1 and Table S2). Collectively, these data indicated that although DNCT expression induced the nuclear localization of TAZ and YAP, it was not sufficient to induce the expression target genes.


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

Ozawa M - Sci Rep (2015)

DNCT expression inhibits YAP/TAZ and β-catenin transcriptional activity.A YAP/TAZ-responsive EGFP reporter (8 × GTIIC-EGFP) (a) or a β-catenin–responsive EGFP reporter (TOP-EGFP) (b) was introduced into DNCT+ cells and stable transfectants were isolated. Cells were cultured for 2 d in the presence (+) or absence (−) of Dox prior to the initiation of the experiments. Then, cells were cultured for 24 h in the presence (+) or absence (−) of LPA (20 μM) or BIO (3 μM) to stimulate YAP/TAZ or β-catenin transcriptional activity, respectively, and were analyzed by flow cytometry.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: DNCT expression inhibits YAP/TAZ and β-catenin transcriptional activity.A YAP/TAZ-responsive EGFP reporter (8 × GTIIC-EGFP) (a) or a β-catenin–responsive EGFP reporter (TOP-EGFP) (b) was introduced into DNCT+ cells and stable transfectants were isolated. Cells were cultured for 2 d in the presence (+) or absence (−) of Dox prior to the initiation of the experiments. Then, cells were cultured for 24 h in the presence (+) or absence (−) of LPA (20 μM) or BIO (3 μM) to stimulate YAP/TAZ or β-catenin transcriptional activity, respectively, and were analyzed by flow cytometry.
Mentions: To determine whether TAZ activity is regulated by DNCT expression, we monitored TAZ transcriptional activity in DNCT+ cells cultured with or without Dox. For this, we used a synthetic YAP/TAZ-responsive EGFP reporter (8×GTIIC-EGFP) as a direct read-out of activity. A similar construct was previously used to successfully monitor TEAD-dependent YAP/TAZ activity24. The present construct was introduced into DNCT+ cells and stable transfectants were isolated. 8×GTIIC-EGFP/DNCT cells cultured in the presence of Dox and without serum exhibited low levels of EGFP protein expression as determined by flow cytometry (Fig. 6a, +Dox/-serum). Lysophosphatidic acid (LPA) has recently shown to activate the YAP/TAZ transcription activity37, and the addition of LPA to cultures increased expression of the EGFP protein (Fig. 6a, +Dox/+LPA). When the cells were cultured in the absence of Dox, EGFP levels were decreased (Fig. 6a, -Dox/-serum), and YAP/TAZ activity was not activated by the addition of LPA (Fig. 6a, -Dox/+LPA). Consistent with these results, a comparison of the gene expression of DNCT+ cells cultured with or without Dox using an Agilent Whole Canine microarray revealed no change in the expression of CTGF (Table S1). Of the 112 transcripts previously identified as significantly increased by YAP overexpression in NIH 3T3 cells19, four were upregulated and three downregulated by DNCT expression; the rest (105 genes) exhibited no change (Table S1 and Table S2). Collectively, these data indicated that although DNCT expression induced the nuclear localization of TAZ and YAP, it was not sufficient to induce the expression target genes.

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