14-3-3ε Overexpression Contributes to Epithelial-Mesenchymal Transition of Hepatocellular Carcinoma

<div><p>Background</p><p>14-3-3ε is implicated in regulating tumor progression, including hepatocellular carcinoma (HCC). Our earlier study indicated that elevated 14-3-3ε expression is significantly associated with higher risk of metastasis and lower survival rates of HCC patients. However, the molecular mechanisms of how 14-3-3ε regulates HCC tumor metastasis are still unclear.</p> <p>Methodology and Principal Findings</p><p>In this study, we show that increased 14-3-3ε expression induces HCC cell migration and promotes epithelial-mesenchymal transition (EMT), which is determined by the reduction of E-cadherin expression and induction of N-cadherin and vimentin expression. Knockdown with specific siRNA abolished 14-3-3ε-induced cell migration and EMT. Furthermore, 14-3-3ε selectively induced Zeb-1 and Snail expression, and 14-3-3ε-induced cell migration was abrogated by Zeb-1 or Snail siRNA. In addition, the effect of 14-3-3ε-reduced E-cadherin was specifically restored by Zeb-1 siRNA. Positive 14-3-3ε expression was significantly correlated with negative E-cadherin expression, as determined by immunohistochemistry analysis in HCC tumors. Analysis of 14-3-3ε/E-cadherin expression associated with clinicopathological characteristics revealed that the combination of positive 14-3-3ε and negative E-cadherin expression is significantly correlated with higher incidence of HCC metastasis and poor 5-year overall survival. In contrast, patients with positive 14-3-3ε and positive E-cadherin expression had better prognostic outcomes than did those with negative E-cadherin expression.</p> <p>Significance</p><p>Our findings show for the first time that E-cadherin is one of the downstream targets of 14-3-3ε in modulating HCC tumor progression. Thus, 14-3-3ε may act as an important regulator in modulating tumor metastasis by promoting EMT as well as cell migration, and it may serve as a novel prognostic biomarker or therapeutic target for HCC.</p> </div

14-3-3ε suppresses E-cadherin expression <i>via</i> regulating Zeb-1.

<p>(A) Cells were transfected with scramble, Snail or/and Zeb-1 siRNAs for 48 hours. E-cadherin, Zeb-1, and Snail protein levels were determined by Western blotting analysis. Actin was used as loading control. (B) E-cadherin expression was determined by quantitative real-time PCR analysis in control and 14-3-3ε overexpression cells. These data are from three independent experiments and presented as the mean ± SD. **<i>P</i><0.01. (C) Expression level and subcellular localization of E-cadherin was examined by immunofluorescent confocal microscopy. (D) 14-3-3ε siRNA dose-dependently decreased Zeb-1/Snail and restore E-cadherin expression in SK-Hep1 cells. Actin was used as loading control.</p

Reversed correlation of 14-3-3ε with E-cadherin expression of hepatocellular carcinoma tumors.

<p>(A) Representative expression of 14-3-3ε and E-cadherin in primary tissue of HCC examined by immunohistochemical analysis. Original magnification, ×200. (B) Significantly reversed correlation of 14-3-3ε with E-cadherin expression in primary HCC tumors was analyzed by Chi-square test.</p

Kaplan-Meier analysis of 14-3-3ε and E-cadherin expression with prognostic outcomes in primary HCC tumors.

<p>E-cadherin positive expression reveals a (A) lower metastatic risk (<i>P</i> = 0.047), and (B) better overall survival rate (<i>P</i> = 0.013) when compared with E-cadherin negative in 14-3-3 positive patients.</p

14-3-3ε promotes epithelial-mesenchymal transition.

<p>(A) Expressions of epithelial and mesenchymal markers included E-cadherin, N-cadherin, and vimentin in control and 14-3-3ε overexpression cells were determined by Western blot analysis and by (B) Immunofluorescent confocal microscopy. (C) 14-3-3ε siRNA-restored E-cadherin and -reduced N-cadherin as well as vimentin expression were determined by Western blotting analysis and by (D) Immunofluorescent confocal microscopy. Actin was used as loading control for protein determination.</p