TNF alpha-Mediated Loss of beta-Catenin/E-Cadherin Association and Subsequent Increase in Cell Migration Is Partially Restored by NKX3.1 Expression in Prostate Cells

WOS: 000343943700018PubMed ID: 25360740Inflammation-induced carcinogenesis is associated with increased proliferation and migration/invasion of various types of tumor cells. In this study, altered beta-catenin signaling upon TNF alpha 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 beta-catenin following increased phosphorylation of Akt((S473)) and GSK3 beta((S9)). Consistently, we observed that subsequent increase in beta-catenin transactivation enhanced c-myc, cyclin D1 and MMP2 expressions. Consequently, it was also observed that the beta-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 beta-catenin by inhibiting Akt((S473)) phosphorylation, therefore, partially rescued the disrupted beta-catenin-E-cadherin interaction as well as the cell migration in LNCaP cells upon cytokine exposure. As, the disrupted localization of beta-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 beta-catenin signaling; consequently sub-cellular localization is deregulated in inflammation, associates with prostatic atrophy and PIN pathology.Turkish Scientific and Technological Research Council (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [108S288, 210T066, 110S134, 113Z083]This research was granted by projects from Turkish Scientific and Technological Research Council (TUBITAK) 108S288 (COST_CANGENIN), 210T066 and 110S134 and 113Z083 to KSK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

High dose (500 pg/ml) of TNFα disrupts LNCaP cell morphology.

<p><b>A.</b> This was examined with regular phase/contrast microscopy (Scale bar represents 50 µm, and the magnification is 10x). <b>B.</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. <b>C.</b> The growth is increased in CM treatments, <b>D.</b> whereas immediate (3 h) and remarkable cell morphology alterations occur in treatments comparison to controls. <b>E.</b> 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.</p

The CM treatment increases cytoplasmic β-catenin accumulation correlating with loss of nuclear NKX3.1 expression.

<p>Tissue sections (containing normal, PIA, H-PIN and PCa regions n = 42, 38, 24 and 24 respectively) were cut from 14 radical prostatectomy specimens and analyzed. The adjacent sections were stained with hematoxylin-eosin dye (<b>A–D</b>), β-catenin (<b>E–H</b>) and NKX3.1 (<b>I–L</b>) antibody to correlate the expression changes in <i>in vivo</i> samples. While glands from the normal prostate exhibited nuclear staining for β-catenin similar to PCa, the atrophy, H-PIN and PCa regions demonstrated remarkable increases in cytoplasmic staining. The representative images were taken from normal glands (<b>A, E, I</b>) the atropy (<b>B, F, J</b>), H-PIN lesions (<b>C, G, K</b>) and prostate adenocarcinoma (<b>D, H, L</b>) samples. The relative intensity from analyzed sections and statistical significance values were also given in comparison to normal sections (<b>M</b>). Histogram plot shows the variation of β-catenin expression between stages (<b>N</b>). The images were taken with a 20× objective. Also, brown colored arrows show the atrophy glands.</p

The CM treatment increases LNCaP cell migration in the Xcelligence CIM-plate.

<p>Additionally, induced migration of the LNCaP cells is positively correlated with the dose of inflammation that was examined using the real-time migration assay. <b>A.</b> 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>B.</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. <b>C.</b> 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.</p

The CM exposure influences prostate cell progression through increasing cytoplasmic accumulation of β-catenin, which subsequently leads increased migration.

<p>Further, inflammation-mediated Akt activity and subsequent β-catenin transactivation can be deregulated by androgen responsive factor NKX3.1, stabilizing the p-β-catenin^(S33) pool, eventually influencing the maintenance of the intact β-catenin/E-cadherin association at the plasma membrane.</p

Inflammation influences the membrane-localized β-catenin and E-cadherin interaction.

<p><b>A.</b> β-catenin and <b>B.</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. <b>C.</b> The loss of membrane-localized β-catenin and E-cadherin interaction at the cell membrane was evidenced when immunoprecipitation (IP) time course was performed. <b>D.</b> 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. <b>E.</b> 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.</p

NKX3.1 suppressed the morphological changes and growth rate of the LNCaP cells, when the cells were treated with CM.

<p><b>A.</b> 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>B.</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. <b>C.</b> 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. <b>D.</b> 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.</p

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

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