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TNFα-mediated loss of β-catenin/E-cadherin association and subsequent increase in cell migration is partially restored by NKX3.1 expression in prostate cells.

Debelec-Butuner B, Alapinar C, Ertunc N, Gonen-Korkmaz C, Yörükoğlu K, Korkmaz KS - PLoS ONE (2014)

Bottom Line: Nevertheless, ectopic expression of NKX3.1, which is degraded upon proinflammatory cytokine exposure in inflammation, was found to induce the degradation of β-catenin by inhibiting Akt(S473) phosphorylation, therefore, partially rescued the disrupted β-catenin-E-cadherin interaction as well as the cell migration in LNCaP cells upon cytokine exposure.As, the disrupted localization of β-catenin at the cell membrane as well as increased Akt(S308) priming phosphorylation was observed in human prostate tissues with prostatic inflammatory atrophy (PIA), high-grade prostatic intraepithelial neoplasia (H-PIN) and carcinoma lesions correlated with loss of NKX3.1 expression.Thus, the data indicate that the β-catenin signaling; consequently sub-cellular localization is deregulated in inflammation, associates with prostatic atrophy and PIN pathology.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Cancer Biology Laboratory, Faculty of Engineering, Ege University, Bornova, Izmir, Turkey; Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Ege University, Bornova, Izmir, Turkey.

ABSTRACT
Inflammation-induced carcinogenesis is associated with increased proliferation and migration/invasion of various types of tumor cells. In this study, altered β-catenin signaling upon TNFα exposure, and relation to loss of function of the tumor suppressor NKX3.1 was examined in prostate cancer cells. We used an in vitro prostate inflammation model to demonstrate altered sub-cellular localization of β-catenin following increased phosphorylation of Akt(S473) and GSK3β(S9). Consistently, we observed that subsequent increase in β-catenin transactivation enhanced c-myc, cyclin D1 and MMP2 expressions. Consequently, it was also observed that the β-catenin-E-cadherin association at the plasma membrane was disrupted during acute cytokine exposure. Additionally, it was demonstrated that disrupting cell-cell interactions led to increased migration of LNCaP cells in real-time migration assay. Nevertheless, ectopic expression of NKX3.1, which is degraded upon proinflammatory cytokine exposure in inflammation, was found to induce the degradation of β-catenin by inhibiting Akt(S473) phosphorylation, therefore, partially rescued the disrupted β-catenin-E-cadherin interaction as well as the cell migration in LNCaP cells upon cytokine exposure. As, the disrupted localization of β-catenin at the cell membrane as well as increased Akt(S308) priming phosphorylation was observed in human prostate tissues with prostatic inflammatory atrophy (PIA), high-grade prostatic intraepithelial neoplasia (H-PIN) and carcinoma lesions correlated with loss of NKX3.1 expression. Thus, the data indicate that the β-catenin signaling; consequently sub-cellular localization is deregulated in inflammation, associates with prostatic atrophy and PIN pathology.

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NKX3.1 suppressed the morphological changes and growth rate of the LNCaP cells, when the cells were treated with CM.A. Significant decreases in p-GSK3β(S9) and p-β-catenin(S552) and an increase in p-β-catenin(S33) phosphorylation were observed after NKX3.1 overexpression. Consistently, c-myc, cyclin D1, and MMP2 expression levels were marginally decreased. B. Although, E-cadherin level is marginally decreased, NKX3.1 expression restored the β-catenin-E-cadherin interaction, which are disrupted by CM (500 pg/ml TNFα for 6 or 24 h) treatment in LNCaP cells. HM-vec and HM-NKX3.1 represent the control and the HM-NKX3.1 expression vectors, respectively. C. LNCaP cells were transfected with the HM control vector or the HM-NKX3.1 for 24 h and the cells were split into E-plates to analyze surface coverage before and after the CM treatments (CM including 250 or 500 pg/ml TNFα for 24 h.). Xcelligence real-time platform was used. The time of 0 indicates when the application of the CM was performed. D. The upregulation of β-catenin is associated with an increase in expression of c-myc and cyclin D1 in chronic CM treatments (62 or 125 pg/ml TNFα for 4 weeks). Consistently, this observation correlates with an increase in p-Akt(S473) level and a decrease in p-β-catenin(S33) in 4 wks of CM treatment. Black arrows indicate that the cellular boundaries of enlarged cells in comparison to control cells (white arrows). Two independent experiments were performed, and the blots were repeated at least three times.
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pone-0109868-g003: NKX3.1 suppressed the morphological changes and growth rate of the LNCaP cells, when the cells were treated with CM.A. Significant decreases in p-GSK3β(S9) and p-β-catenin(S552) and an increase in p-β-catenin(S33) phosphorylation were observed after NKX3.1 overexpression. Consistently, c-myc, cyclin D1, and MMP2 expression levels were marginally decreased. B. Although, E-cadherin level is marginally decreased, NKX3.1 expression restored the β-catenin-E-cadherin interaction, which are disrupted by CM (500 pg/ml TNFα for 6 or 24 h) treatment in LNCaP cells. HM-vec and HM-NKX3.1 represent the control and the HM-NKX3.1 expression vectors, respectively. C. LNCaP cells were transfected with the HM control vector or the HM-NKX3.1 for 24 h and the cells were split into E-plates to analyze surface coverage before and after the CM treatments (CM including 250 or 500 pg/ml TNFα for 24 h.). Xcelligence real-time platform was used. The time of 0 indicates when the application of the CM was performed. D. The upregulation of β-catenin is associated with an increase in expression of c-myc and cyclin D1 in chronic CM treatments (62 or 125 pg/ml TNFα for 4 weeks). Consistently, this observation correlates with an increase in p-Akt(S473) level and a decrease in p-β-catenin(S33) in 4 wks of CM treatment. Black arrows indicate that the cellular boundaries of enlarged cells in comparison to control cells (white arrows). Two independent experiments were performed, and the blots were repeated at least three times.

Mentions: NKX3.1 is a component of transcriptional repressor complex Groucho, which represses TCF4/β-catenin transcriptional activity [23], and is known as an oxidative damage regulator in prostate [17], [18]. Therefore, we examined putative role of NKX3.1 in β-catenin signaling after CM treatment (including 500 pg/ml TNFα, for 6 or 24 h). We found that ectopic NKX3.1 expression reduced the overall stability of β-catenin by suppressing Akt(S473) phosphorylation and consistently restored GSK3β(S9) and β-catenin(S33) but not β-catenin(S552) phosphorylation. Subsequently, the β-catenin transcriptional targets c-myc, cyclin D1 and MMP2 were downregulated both in the control and in the 6 h CM-treated samples (Figure 3A). Furthermore, immunoprecipitation of β-catenin followed by immunoblotting for E-cadherin showed that the β-catenin-E-cadherin association was partially restored both in the control and 6 h CM-treated cells expressing NKX3.1 (Figure 3B). To determine the regulatory role of NKX3.1, cell growth was also examined using the real-time assay in LNCaP cells after treatment with CM including two doses of TNFα (250 or 500 pg/ml). In cells expressing NKX3.1, the CM-mediated morphological changes were reversed remarkably and the cell growth was suppressed even under CM treatments (Figure 3C). These results imply that NKX3.1 has important functions in regulating cell morphology – perhaps taking a suppressive role on the epithelial-mesenchymal transition – and growth that are abrogated under inflammatory like conditions. Thus, NKX3.1 has an important function in controlling cell growth by regulating the β-catenin signaling and partially maintains plasma membrane localization of β-catenin at normal boundaries thereby stabilizing the β-catenin-E-cadherin association.


TNFα-mediated loss of β-catenin/E-cadherin association and subsequent increase in cell migration is partially restored by NKX3.1 expression in prostate cells.

Debelec-Butuner B, Alapinar C, Ertunc N, Gonen-Korkmaz C, Yörükoğlu K, Korkmaz KS - PLoS ONE (2014)

NKX3.1 suppressed the morphological changes and growth rate of the LNCaP cells, when the cells were treated with CM.A. Significant decreases in p-GSK3β(S9) and p-β-catenin(S552) and an increase in p-β-catenin(S33) phosphorylation were observed after NKX3.1 overexpression. Consistently, c-myc, cyclin D1, and MMP2 expression levels were marginally decreased. B. Although, E-cadherin level is marginally decreased, NKX3.1 expression restored the β-catenin-E-cadherin interaction, which are disrupted by CM (500 pg/ml TNFα for 6 or 24 h) treatment in LNCaP cells. HM-vec and HM-NKX3.1 represent the control and the HM-NKX3.1 expression vectors, respectively. C. LNCaP cells were transfected with the HM control vector or the HM-NKX3.1 for 24 h and the cells were split into E-plates to analyze surface coverage before and after the CM treatments (CM including 250 or 500 pg/ml TNFα for 24 h.). Xcelligence real-time platform was used. The time of 0 indicates when the application of the CM was performed. D. The upregulation of β-catenin is associated with an increase in expression of c-myc and cyclin D1 in chronic CM treatments (62 or 125 pg/ml TNFα for 4 weeks). Consistently, this observation correlates with an increase in p-Akt(S473) level and a decrease in p-β-catenin(S33) in 4 wks of CM treatment. Black arrows indicate that the cellular boundaries of enlarged cells in comparison to control cells (white arrows). Two independent experiments were performed, and the blots were repeated at least three times.
© Copyright Policy
Related In: Results  -  Collection

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pone-0109868-g003: NKX3.1 suppressed the morphological changes and growth rate of the LNCaP cells, when the cells were treated with CM.A. Significant decreases in p-GSK3β(S9) and p-β-catenin(S552) and an increase in p-β-catenin(S33) phosphorylation were observed after NKX3.1 overexpression. Consistently, c-myc, cyclin D1, and MMP2 expression levels were marginally decreased. B. Although, E-cadherin level is marginally decreased, NKX3.1 expression restored the β-catenin-E-cadherin interaction, which are disrupted by CM (500 pg/ml TNFα for 6 or 24 h) treatment in LNCaP cells. HM-vec and HM-NKX3.1 represent the control and the HM-NKX3.1 expression vectors, respectively. C. LNCaP cells were transfected with the HM control vector or the HM-NKX3.1 for 24 h and the cells were split into E-plates to analyze surface coverage before and after the CM treatments (CM including 250 or 500 pg/ml TNFα for 24 h.). Xcelligence real-time platform was used. The time of 0 indicates when the application of the CM was performed. D. The upregulation of β-catenin is associated with an increase in expression of c-myc and cyclin D1 in chronic CM treatments (62 or 125 pg/ml TNFα for 4 weeks). Consistently, this observation correlates with an increase in p-Akt(S473) level and a decrease in p-β-catenin(S33) in 4 wks of CM treatment. Black arrows indicate that the cellular boundaries of enlarged cells in comparison to control cells (white arrows). Two independent experiments were performed, and the blots were repeated at least three times.
Mentions: NKX3.1 is a component of transcriptional repressor complex Groucho, which represses TCF4/β-catenin transcriptional activity [23], and is known as an oxidative damage regulator in prostate [17], [18]. Therefore, we examined putative role of NKX3.1 in β-catenin signaling after CM treatment (including 500 pg/ml TNFα, for 6 or 24 h). We found that ectopic NKX3.1 expression reduced the overall stability of β-catenin by suppressing Akt(S473) phosphorylation and consistently restored GSK3β(S9) and β-catenin(S33) but not β-catenin(S552) phosphorylation. Subsequently, the β-catenin transcriptional targets c-myc, cyclin D1 and MMP2 were downregulated both in the control and in the 6 h CM-treated samples (Figure 3A). Furthermore, immunoprecipitation of β-catenin followed by immunoblotting for E-cadherin showed that the β-catenin-E-cadherin association was partially restored both in the control and 6 h CM-treated cells expressing NKX3.1 (Figure 3B). To determine the regulatory role of NKX3.1, cell growth was also examined using the real-time assay in LNCaP cells after treatment with CM including two doses of TNFα (250 or 500 pg/ml). In cells expressing NKX3.1, the CM-mediated morphological changes were reversed remarkably and the cell growth was suppressed even under CM treatments (Figure 3C). These results imply that NKX3.1 has important functions in regulating cell morphology – perhaps taking a suppressive role on the epithelial-mesenchymal transition – and growth that are abrogated under inflammatory like conditions. Thus, NKX3.1 has an important function in controlling cell growth by regulating the β-catenin signaling and partially maintains plasma membrane localization of β-catenin at normal boundaries thereby stabilizing the β-catenin-E-cadherin association.

Bottom Line: Nevertheless, ectopic expression of NKX3.1, which is degraded upon proinflammatory cytokine exposure in inflammation, was found to induce the degradation of β-catenin by inhibiting Akt(S473) phosphorylation, therefore, partially rescued the disrupted β-catenin-E-cadherin interaction as well as the cell migration in LNCaP cells upon cytokine exposure.As, the disrupted localization of β-catenin at the cell membrane as well as increased Akt(S308) priming phosphorylation was observed in human prostate tissues with prostatic inflammatory atrophy (PIA), high-grade prostatic intraepithelial neoplasia (H-PIN) and carcinoma lesions correlated with loss of NKX3.1 expression.Thus, the data indicate that the β-catenin signaling; consequently sub-cellular localization is deregulated in inflammation, associates with prostatic atrophy and PIN pathology.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Cancer Biology Laboratory, Faculty of Engineering, Ege University, Bornova, Izmir, Turkey; Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Ege University, Bornova, Izmir, Turkey.

ABSTRACT
Inflammation-induced carcinogenesis is associated with increased proliferation and migration/invasion of various types of tumor cells. In this study, altered β-catenin signaling upon TNFα exposure, and relation to loss of function of the tumor suppressor NKX3.1 was examined in prostate cancer cells. We used an in vitro prostate inflammation model to demonstrate altered sub-cellular localization of β-catenin following increased phosphorylation of Akt(S473) and GSK3β(S9). Consistently, we observed that subsequent increase in β-catenin transactivation enhanced c-myc, cyclin D1 and MMP2 expressions. Consequently, it was also observed that the β-catenin-E-cadherin association at the plasma membrane was disrupted during acute cytokine exposure. Additionally, it was demonstrated that disrupting cell-cell interactions led to increased migration of LNCaP cells in real-time migration assay. Nevertheless, ectopic expression of NKX3.1, which is degraded upon proinflammatory cytokine exposure in inflammation, was found to induce the degradation of β-catenin by inhibiting Akt(S473) phosphorylation, therefore, partially rescued the disrupted β-catenin-E-cadherin interaction as well as the cell migration in LNCaP cells upon cytokine exposure. As, the disrupted localization of β-catenin at the cell membrane as well as increased Akt(S308) priming phosphorylation was observed in human prostate tissues with prostatic inflammatory atrophy (PIA), high-grade prostatic intraepithelial neoplasia (H-PIN) and carcinoma lesions correlated with loss of NKX3.1 expression. Thus, the data indicate that the β-catenin signaling; consequently sub-cellular localization is deregulated in inflammation, associates with prostatic atrophy and PIN pathology.

Show MeSH
Related in: MedlinePlus