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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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High dose (500 pg/ml) of TNFα disrupts LNCaP cell morphology.A. This was examined with regular phase/contrast microscopy (Scale bar represents 50 µm, and the magnification is 10x). B. When LNCaP cells were examined using a real-time cell proliferation assay, 3 to 6 h after the CM treatment (500 pg/ml TNFα), significant (p<0.001) augmentation of the cell surface area was observed. Real-time cell proliferation assay was performed twice in each 6 identical replicates and the western blots were performed at least twice as independent replicates. C. The growth is increased in CM treatments, D. whereas immediate (3 h) and remarkable cell morphology alterations occur in treatments comparison to controls. E. In CM treatment, β-catenin expression is upregulated besides increased Akt(S473) phosphorylation. Also, inhibitory phosphorylation of GSK3β(S9) and the stability-enhancing phosphorylation of β-catenin(S552) increased concurrently to the p-β-catenin(S33) decrease. As a result, the stabilized β-catenin-mediated transactivation increased the expression of c-myc, cyclin D1 and MMP2 that are shown.
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pone-0109868-g001: High dose (500 pg/ml) of TNFα disrupts LNCaP cell morphology.A. This was examined with regular phase/contrast microscopy (Scale bar represents 50 µm, and the magnification is 10x). B. When LNCaP cells were examined using a real-time cell proliferation assay, 3 to 6 h after the CM treatment (500 pg/ml TNFα), significant (p<0.001) augmentation of the cell surface area was observed. Real-time cell proliferation assay was performed twice in each 6 identical replicates and the western blots were performed at least twice as independent replicates. C. The growth is increased in CM treatments, D. whereas immediate (3 h) and remarkable cell morphology alterations occur in treatments comparison to controls. E. In CM treatment, β-catenin expression is upregulated besides increased Akt(S473) phosphorylation. Also, inhibitory phosphorylation of GSK3β(S9) and the stability-enhancing phosphorylation of β-catenin(S552) increased concurrently to the p-β-catenin(S33) decrease. As a result, the stabilized β-catenin-mediated transactivation increased the expression of c-myc, cyclin D1 and MMP2 that are shown.

Mentions: To understand the role of the inflammatory microenvironment in prostate cells, optimized conditioned media (CM) including TNFα was added onto the LNCaP cells [20], and then the cellular alterations, cell surface coverage as well as growth were examined. At certain doses (250 and 500 pg/ml TNFα adjusted) and specific time points (3–6 h) of the CM treatments, an immediate and considerable response was observed in prostate cells (Figure 1A). To further characterize this response, cell growth was examined using a real-time proliferation assay, and the impedance readings from the growing cultures were converted into growth rate using the Xcelligence system. First, LNCaP cells were propagated for 24 h in a resting state, and then fed with CM. The impedance values were recorded real-time over 10 min intervals from each well. Thus, the treatments significantly changed the impedance corresponding to cell growth within 3 h (p<0.001) compared to untreated controls (Figure 1B). Nevertheless, as the observed rate of change (2.4-fold in 3 h) could not be due to the cellular growth and immediately occurred after CM treatment, we assumed that this could be a change in surface coverage of the cells and performed WST based assay measuring mitochondrial ATPase production (Figure 1C). The data suggest that an immediate change in cell morphology occurs upon CM exposure concurrent the increased cell growth (Figure 1D).


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)

High dose (500 pg/ml) of TNFα disrupts LNCaP cell morphology.A. This was examined with regular phase/contrast microscopy (Scale bar represents 50 µm, and the magnification is 10x). B. When LNCaP cells were examined using a real-time cell proliferation assay, 3 to 6 h after the CM treatment (500 pg/ml TNFα), significant (p<0.001) augmentation of the cell surface area was observed. Real-time cell proliferation assay was performed twice in each 6 identical replicates and the western blots were performed at least twice as independent replicates. C. The growth is increased in CM treatments, D. whereas immediate (3 h) and remarkable cell morphology alterations occur in treatments comparison to controls. E. In CM treatment, β-catenin expression is upregulated besides increased Akt(S473) phosphorylation. Also, inhibitory phosphorylation of GSK3β(S9) and the stability-enhancing phosphorylation of β-catenin(S552) increased concurrently to the p-β-catenin(S33) decrease. As a result, the stabilized β-catenin-mediated transactivation increased the expression of c-myc, cyclin D1 and MMP2 that are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0109868-g001: High dose (500 pg/ml) of TNFα disrupts LNCaP cell morphology.A. This was examined with regular phase/contrast microscopy (Scale bar represents 50 µm, and the magnification is 10x). B. When LNCaP cells were examined using a real-time cell proliferation assay, 3 to 6 h after the CM treatment (500 pg/ml TNFα), significant (p<0.001) augmentation of the cell surface area was observed. Real-time cell proliferation assay was performed twice in each 6 identical replicates and the western blots were performed at least twice as independent replicates. C. The growth is increased in CM treatments, D. whereas immediate (3 h) and remarkable cell morphology alterations occur in treatments comparison to controls. E. In CM treatment, β-catenin expression is upregulated besides increased Akt(S473) phosphorylation. Also, inhibitory phosphorylation of GSK3β(S9) and the stability-enhancing phosphorylation of β-catenin(S552) increased concurrently to the p-β-catenin(S33) decrease. As a result, the stabilized β-catenin-mediated transactivation increased the expression of c-myc, cyclin D1 and MMP2 that are shown.
Mentions: To understand the role of the inflammatory microenvironment in prostate cells, optimized conditioned media (CM) including TNFα was added onto the LNCaP cells [20], and then the cellular alterations, cell surface coverage as well as growth were examined. At certain doses (250 and 500 pg/ml TNFα adjusted) and specific time points (3–6 h) of the CM treatments, an immediate and considerable response was observed in prostate cells (Figure 1A). To further characterize this response, cell growth was examined using a real-time proliferation assay, and the impedance readings from the growing cultures were converted into growth rate using the Xcelligence system. First, LNCaP cells were propagated for 24 h in a resting state, and then fed with CM. The impedance values were recorded real-time over 10 min intervals from each well. Thus, the treatments significantly changed the impedance corresponding to cell growth within 3 h (p<0.001) compared to untreated controls (Figure 1B). Nevertheless, as the observed rate of change (2.4-fold in 3 h) could not be due to the cellular growth and immediately occurred after CM treatment, we assumed that this could be a change in surface coverage of the cells and performed WST based assay measuring mitochondrial ATPase production (Figure 1C). The data suggest that an immediate change in cell morphology occurs upon CM exposure concurrent the increased cell growth (Figure 1D).

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