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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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The CM treatment increases LNCaP cell migration in the Xcelligence CIM-plate.Additionally, induced migration of the LNCaP cells is positively correlated with the dose of inflammation that was examined using the real-time migration assay. A. N2: Negative control; medium containing 2% FBS was placed in both the upper and lower chambers. N10: Chemo-attractant control; medium containing 2% FBS was placed in the upper chamber, and medium containing 10% FBS was placed in the lower chamber of the CIM-plate. B. Ectopic NKX3.1 expression suppresses the inflammatory microenvironment-mediated migration of the LNCaP cells (green line). HM-Vec and HM-NKX3.1 indicate the control and the HisMax-NKX3.1 expression vectors, respectively. C. The cells exhibit clear membrane-localized β-catenin (upper panel). Although, membrane localized β-catenin level remains higher in cells that are expressing NKX3.1 at high levels, the cells responded to CM (250 pg/ml for 3 h) treatments and promoted the variable expression/localization of β-catenin correlated with remarkably variable NKX3.1 expression (lower panel). Blue dashed line indicates the region with depleted NKX3.1 expression, where β-catenin is also decreased especially at membrane boundaries.
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pone-0109868-g004: The CM treatment increases LNCaP cell migration in the Xcelligence CIM-plate.Additionally, induced migration of the LNCaP cells is positively correlated with the dose of inflammation that was examined using the real-time migration assay. A. N2: Negative control; medium containing 2% FBS was placed in both the upper and lower chambers. N10: Chemo-attractant control; medium containing 2% FBS was placed in the upper chamber, and medium containing 10% FBS was placed in the lower chamber of the CIM-plate. B. Ectopic NKX3.1 expression suppresses the inflammatory microenvironment-mediated migration of the LNCaP cells (green line). HM-Vec and HM-NKX3.1 indicate the control and the HisMax-NKX3.1 expression vectors, respectively. C. The cells exhibit clear membrane-localized β-catenin (upper panel). Although, membrane localized β-catenin level remains higher in cells that are expressing NKX3.1 at high levels, the cells responded to CM (250 pg/ml for 3 h) treatments and promoted the variable expression/localization of β-catenin correlated with remarkably variable NKX3.1 expression (lower panel). Blue dashed line indicates the region with depleted NKX3.1 expression, where β-catenin is also decreased especially at membrane boundaries.

Mentions: To study the migration of LNCaP cells real-time Boyden chamber based migration assay was used (Xcelligence system). In this assay, FBS was used as a chemo-attractant, and 10% normal serum (N10) was used for positive control. We then used 2% FBS (N2) in the upper chamber of the CIM plate. As a negative control, 2% FBS (N2) was placed into both chambers. When the LNCaP cells were treated with the CM (including 250 or 500 pg/ml TNFα for 3 h and split onto the CIM-plate) the migration ability of the cells were correlated with increasing CM doses significantly (p<0.001) (Figure 4A). Additionally, the role of NKX3.1 expression in cell migration was examined and found that NKX3.1 significantly (p<0.001) suppressed the CM-induced migration of the LNCaP cells (Figure 4B). In order to understand how β-catenin stabilization correlates with NKX3.1 expression level, we also treated cells with 250 pg/ml TNFα containing CM that is enough for NKX3.1 degradation, and co-stained with β-catenin and NKX3.1. The data revealed that β-catenin stabilizes at membrane localizations with ectopic NKX3.1 expression and is disrupted with CM, concurrent to loss of NKX3.1 expression (Figure 4C). Thus, the data suggested that the NKX3.1 is an important factor for β-catenin localizations upon inflammation.


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)

The CM treatment increases LNCaP cell migration in the Xcelligence CIM-plate.Additionally, induced migration of the LNCaP cells is positively correlated with the dose of inflammation that was examined using the real-time migration assay. A. N2: Negative control; medium containing 2% FBS was placed in both the upper and lower chambers. N10: Chemo-attractant control; medium containing 2% FBS was placed in the upper chamber, and medium containing 10% FBS was placed in the lower chamber of the CIM-plate. B. Ectopic NKX3.1 expression suppresses the inflammatory microenvironment-mediated migration of the LNCaP cells (green line). HM-Vec and HM-NKX3.1 indicate the control and the HisMax-NKX3.1 expression vectors, respectively. C. The cells exhibit clear membrane-localized β-catenin (upper panel). Although, membrane localized β-catenin level remains higher in cells that are expressing NKX3.1 at high levels, the cells responded to CM (250 pg/ml for 3 h) treatments and promoted the variable expression/localization of β-catenin correlated with remarkably variable NKX3.1 expression (lower panel). Blue dashed line indicates the region with depleted NKX3.1 expression, where β-catenin is also decreased especially at membrane boundaries.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4215977&req=5

pone-0109868-g004: The CM treatment increases LNCaP cell migration in the Xcelligence CIM-plate.Additionally, induced migration of the LNCaP cells is positively correlated with the dose of inflammation that was examined using the real-time migration assay. A. N2: Negative control; medium containing 2% FBS was placed in both the upper and lower chambers. N10: Chemo-attractant control; medium containing 2% FBS was placed in the upper chamber, and medium containing 10% FBS was placed in the lower chamber of the CIM-plate. B. Ectopic NKX3.1 expression suppresses the inflammatory microenvironment-mediated migration of the LNCaP cells (green line). HM-Vec and HM-NKX3.1 indicate the control and the HisMax-NKX3.1 expression vectors, respectively. C. The cells exhibit clear membrane-localized β-catenin (upper panel). Although, membrane localized β-catenin level remains higher in cells that are expressing NKX3.1 at high levels, the cells responded to CM (250 pg/ml for 3 h) treatments and promoted the variable expression/localization of β-catenin correlated with remarkably variable NKX3.1 expression (lower panel). Blue dashed line indicates the region with depleted NKX3.1 expression, where β-catenin is also decreased especially at membrane boundaries.
Mentions: To study the migration of LNCaP cells real-time Boyden chamber based migration assay was used (Xcelligence system). In this assay, FBS was used as a chemo-attractant, and 10% normal serum (N10) was used for positive control. We then used 2% FBS (N2) in the upper chamber of the CIM plate. As a negative control, 2% FBS (N2) was placed into both chambers. When the LNCaP cells were treated with the CM (including 250 or 500 pg/ml TNFα for 3 h and split onto the CIM-plate) the migration ability of the cells were correlated with increasing CM doses significantly (p<0.001) (Figure 4A). Additionally, the role of NKX3.1 expression in cell migration was examined and found that NKX3.1 significantly (p<0.001) suppressed the CM-induced migration of the LNCaP cells (Figure 4B). In order to understand how β-catenin stabilization correlates with NKX3.1 expression level, we also treated cells with 250 pg/ml TNFα containing CM that is enough for NKX3.1 degradation, and co-stained with β-catenin and NKX3.1. The data revealed that β-catenin stabilizes at membrane localizations with ectopic NKX3.1 expression and is disrupted with CM, concurrent to loss of NKX3.1 expression (Figure 4C). Thus, the data suggested that the NKX3.1 is an important factor for β-catenin localizations upon inflammation.

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