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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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Inflammation influences the membrane-localized β-catenin and E-cadherin interaction.A. β-catenin and B. p-β-catenin(S552) localizations at plasma membrane (arrows) are decreased with CM treatment (500 pg/ml TNFα for 3 h) (magnification 60X), and the β-catenin and p-β-catenin(S552) localize into cell cytoplasm. C. The loss of membrane-localized β-catenin and E-cadherin interaction at the cell membrane was evidenced when immunoprecipitation (IP) time course was performed. D. A substantial increase in cytoplasmic and nuclear translocated β-catenin after CM treatments were also confirmed in the sub-cellular fractionated cell lysates. E-cadherin, H2A and GAPDH levels were also confirmed not changing after 6 h of treatment as controls for fractions. Memb: Membrane fraction, Nuc: Nuclear fraction, Cyto: Cytoplasmic fraction. E. Furthermore, total ubiquitination as well as β-catenin expression levels are increased in CM treated LNCaP cells whereas β-catenin ubiquitination is decreased. The antibodies for IPs were anti-mouse IgG or NKX3.1 and/or anti-β-catenin. The IPs and the blots were performed at least three times.
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pone-0109868-g002: Inflammation influences the membrane-localized β-catenin and E-cadherin interaction.A. β-catenin and B. p-β-catenin(S552) localizations at plasma membrane (arrows) are decreased with CM treatment (500 pg/ml TNFα for 3 h) (magnification 60X), and the β-catenin and p-β-catenin(S552) localize into cell cytoplasm. C. The loss of membrane-localized β-catenin and E-cadherin interaction at the cell membrane was evidenced when immunoprecipitation (IP) time course was performed. D. A substantial increase in cytoplasmic and nuclear translocated β-catenin after CM treatments were also confirmed in the sub-cellular fractionated cell lysates. E-cadherin, H2A and GAPDH levels were also confirmed not changing after 6 h of treatment as controls for fractions. Memb: Membrane fraction, Nuc: Nuclear fraction, Cyto: Cytoplasmic fraction. E. Furthermore, total ubiquitination as well as β-catenin expression levels are increased in CM treated LNCaP cells whereas β-catenin ubiquitination is decreased. The antibodies for IPs were anti-mouse IgG or NKX3.1 and/or anti-β-catenin. The IPs and the blots were performed at least three times.

Mentions: To determine the influence of inflammation in terms of β-catenin stabilization, subcellular localization of β-catenin, degree of phosphorylation at S552 residue and the interaction between β-catenin and E-cadherin were investigated using immunofluorescence microscopy and immunoprecipitation studies. First of all, we observed that the localization of β-catenin at membrane localizations was disrupted in LNCaP cells when they were treated with 500 pg/ml TNFα containing CM for 3 to 6 h (Figure 2A). Additionally, p-β-catenin(S552) was increased at cytoplasm at 3 h of CM treatment (Figure 2B). Although the total E-cadherin and β-catenin expression levels remained similar in treatments (inputs in Figure 2C), we found an immediate and substantial loss in β-catenin-E-cadherin interaction after 3 h CM treatment (Figure 2C). To validate the increased transactivation of β-catenin by its altered subcellular localization, nuclear, cytoplasmic and membrane proteins were fractionated and β-catenin, E-cadherin, cyclin D1 and control proteins GAPDH and Histone 2 A (H2A) expressions were examined before and after CM treatments. Subcellular fractionation coupled western blotting revealed that β-catenin level increased substantially in cytoplasmic fraction after 6 h of CM treatment relative to GAPDH expression (Figure 2D). Consequently, β-catenin transactivation increased, which was confirmed via cyclin D1 expression (Figure 2D), though GAPDH, H2A and E-cadherin levels remained same. Finally, β-catenin ubiquitination was analyzed using IPs and displayed that β-catenin expression inversely correlated with ubiqitination of β-catenin in CM treated cells. Taken together, the data suggest that the β-catenin stabilization occurs due to inhibition of β-catenin degradation (Figure 2E).


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)

Inflammation influences the membrane-localized β-catenin and E-cadherin interaction.A. β-catenin and B. p-β-catenin(S552) localizations at plasma membrane (arrows) are decreased with CM treatment (500 pg/ml TNFα for 3 h) (magnification 60X), and the β-catenin and p-β-catenin(S552) localize into cell cytoplasm. C. The loss of membrane-localized β-catenin and E-cadherin interaction at the cell membrane was evidenced when immunoprecipitation (IP) time course was performed. D. A substantial increase in cytoplasmic and nuclear translocated β-catenin after CM treatments were also confirmed in the sub-cellular fractionated cell lysates. E-cadherin, H2A and GAPDH levels were also confirmed not changing after 6 h of treatment as controls for fractions. Memb: Membrane fraction, Nuc: Nuclear fraction, Cyto: Cytoplasmic fraction. E. Furthermore, total ubiquitination as well as β-catenin expression levels are increased in CM treated LNCaP cells whereas β-catenin ubiquitination is decreased. The antibodies for IPs were anti-mouse IgG or NKX3.1 and/or anti-β-catenin. The IPs and the blots were performed at least three times.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4215977&req=5

pone-0109868-g002: Inflammation influences the membrane-localized β-catenin and E-cadherin interaction.A. β-catenin and B. p-β-catenin(S552) localizations at plasma membrane (arrows) are decreased with CM treatment (500 pg/ml TNFα for 3 h) (magnification 60X), and the β-catenin and p-β-catenin(S552) localize into cell cytoplasm. C. The loss of membrane-localized β-catenin and E-cadherin interaction at the cell membrane was evidenced when immunoprecipitation (IP) time course was performed. D. A substantial increase in cytoplasmic and nuclear translocated β-catenin after CM treatments were also confirmed in the sub-cellular fractionated cell lysates. E-cadherin, H2A and GAPDH levels were also confirmed not changing after 6 h of treatment as controls for fractions. Memb: Membrane fraction, Nuc: Nuclear fraction, Cyto: Cytoplasmic fraction. E. Furthermore, total ubiquitination as well as β-catenin expression levels are increased in CM treated LNCaP cells whereas β-catenin ubiquitination is decreased. The antibodies for IPs were anti-mouse IgG or NKX3.1 and/or anti-β-catenin. The IPs and the blots were performed at least three times.
Mentions: To determine the influence of inflammation in terms of β-catenin stabilization, subcellular localization of β-catenin, degree of phosphorylation at S552 residue and the interaction between β-catenin and E-cadherin were investigated using immunofluorescence microscopy and immunoprecipitation studies. First of all, we observed that the localization of β-catenin at membrane localizations was disrupted in LNCaP cells when they were treated with 500 pg/ml TNFα containing CM for 3 to 6 h (Figure 2A). Additionally, p-β-catenin(S552) was increased at cytoplasm at 3 h of CM treatment (Figure 2B). Although the total E-cadherin and β-catenin expression levels remained similar in treatments (inputs in Figure 2C), we found an immediate and substantial loss in β-catenin-E-cadherin interaction after 3 h CM treatment (Figure 2C). To validate the increased transactivation of β-catenin by its altered subcellular localization, nuclear, cytoplasmic and membrane proteins were fractionated and β-catenin, E-cadherin, cyclin D1 and control proteins GAPDH and Histone 2 A (H2A) expressions were examined before and after CM treatments. Subcellular fractionation coupled western blotting revealed that β-catenin level increased substantially in cytoplasmic fraction after 6 h of CM treatment relative to GAPDH expression (Figure 2D). Consequently, β-catenin transactivation increased, which was confirmed via cyclin D1 expression (Figure 2D), though GAPDH, H2A and E-cadherin levels remained same. Finally, β-catenin ubiquitination was analyzed using IPs and displayed that β-catenin expression inversely correlated with ubiqitination of β-catenin in CM treated cells. Taken together, the data suggest that the β-catenin stabilization occurs due to inhibition of β-catenin degradation (Figure 2E).

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