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. 2017 Jun;108(6):1157-1168.
doi: 10.1111/cas.13247. Epub 2017 Jun 13.

Downregulation of Talin1 promotes hepatocellular carcinoma progression through activation of the ERK1/2 pathway

Peijuan Chen  1 , Ling Lei  1 , Jian Wang  1 , Xuejing Zou  2 , Dongyan Zhang  1 , Ling Deng  1 , Dehua Wu  1
Affiliations

Downregulation of Talin1 promotes hepatocellular carcinoma progression through activation of the ERK1/2 pathway

Peijuan Chen et al. Cancer Sci. 2017 Jun.

Abstract

Talin1 is an adaptor protein that conjugates integrins to the cytoskeleton and regulates integrins and focal adhesion signaling. Several studies have found that Talin1 is overexpressed in several tumor types and promotes tumor progression. However, the explicit role of Talin1 in hepatocellular carcinoma (HCC) progression is still unclear and its functional mechanism remains largely unknown. In this study, we showed a trend of gradually decreasing expression of Talin1 from normal liver tissues to hepatocirrhosis, liver hyperplasia, the corresponding adjacent non-tumor, primary HCC, and eventually metastatic foci, indicating that Talin1 may correlate with HCC initiation to progression. Talin1 was significantly downregulated in HCC tissues compared with adjacent non-tumor tissues and low Talin1 expression was associated with HCC progression and poor prognosis. Furthermore, Talin1 knockdown induced epithelial-mesenchymal transition and promoted migration and invasion in SK-Hep-1 cells and HepG2 cells. Mechanistically, we found that the ERK pathway was responsible for these promoting effects of Talin1 knockdown in HCC cells. The promoting effects of Talin1 knockdown on epithelial-mesenchymal transition, migration, and invasion were reversed by U0126, a specific ERK1/2 inhibitor. Taken together, our results suggested that Talin1 might serve as a tumor suppressor in HCC and a potential prognostic biomarker for HCC patients.

Keywords: ERK1/2 pathway; Epithelial-mesenchymal transition; Talin1; hepatocellular carcinoma; prognostic biomarker.

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Figures

Figure 1
Figure 1
Talin1 is significantly down‐regulated in HCC tissues. (a) Talin1 expression in indicated tissues was determined by IHC assays (*P < 0.05, **P < 0.01). (b) Representative images of Talin1 expression from normal liver tissues, cirrhosis tissues, hyperplasia tissues, corresponding adjacent non‐tumor tissues, primary tumor tissues, and metastasis #1 (left index finger), metastasis #3 (distal stomach), and metastasis #7 (the top of the brain) by IHC assays. (c) qRT‐PCR analysis of relative Talin1 mRNA level in 36 HCC tissues and the corresponding non‐tumor liver tissues after normalization for the endogenous control (β‐actin). (d) Quantification of Talin1 mRNA expression in HCC tissues and non‐tumor tissues (***P < 0.001). (e) Western blot analysis of Talin1 expression in HCC tissues (T) and corresponding adjacent non‐tumor tissues (N) (n = 12).
Figure 2
Figure 2
Low Talin1 expression correlates with poor prognosis of hepatocellular carcinoma (HCC) patients. (a) Representative images of Talin1 expression from non‐tumor tissues and HCC tissues by immunohistochemical assays. Scale bar = 50 μm (right panels), 100 μm (left panels). (b) Talin1 expression in non‐tumor tissues (n = 196) and HCC tissues (n = 200) was determined by immunohistochemistry. ***P < 0.001. (c,d) Kaplan–Meier and log–rank test analyses of overall survival (c) and disease‐free survival (d) in 200 HCC patients based on Talin1 expression level of HCC tissues. **P < 0.01; ***P < 0.001.
Figure 3
Figure 3
Knockdown of Talin1 induces epithelial–mesenchymal transition in hepatocellular carcinoma (HCC) cells. (a) Talin1 expression was assessed in indicated HCC cell lines by Western blot analysis. (b) Western blot analysis showed knockdown of Talin1 protein in SK‐Hep‐1 and HepG2 cells transfected with Talin1‐specific siRNA#1, #2, #3, or #4. Talin1 was quantified as integrated density to represent the amount of protein. Numerical values represented the quantified data as normalized to the scramble group. (c) SK‐Hep‐1 and HepG2 cells transfected with scrambled or Talin1‐specific siRNA are shown by phase contrast microscopy. Original magnification, ×400. (d) Western blot analysis for the expression of Talin1, E‐cadherin, N‐cadherin, vimentin, and β‐catenin in Talin1‐silenced SK‐Hep‐1 and HepG2 cells. (e) Correlation between Talin1 and E‐cadherin protein expression was detected in 12 pairs of HCC tissues and corresponding adjacent non‐tumor tissues by Western blot analysis. Spearman's rank correlation, r = 0.622; P = 0.031. (f) Immunofluorescence staining showed that knockdown of Talin1 altered the subcellular redistribution of β‐catenin. Blue, DAPI; red, β‐catenin antibody.
Figure 4
Figure 4
Knockdown of Talin1 promotes hepatocellular carcinoma cell migration and invasion. (a,b) Representative images of Transwell migration assays and matrix‐coated Transwell invasion assays in SK‐Hep‐1 and HepG2 cells from indicated groups (left panels). Bar charts represent the average number of migratory cells and invaded cells from five random microscopic fields. *P < 0.05; **P < 0.01; ***P < 0.001. (c) FITC‐phalloidin staining determined the effect of Talin1 knockdown on the growth of lamellipodia in SK‐Hep‐1 cells.
Figure 5
Figure 5
The ERK1/2 pathway is involved in Talin1 knockdown‐induced epithelial–mesenchymal transition effects in hepatocellular carcinoma (HCC) cells. (a) Expression of phosphorylated focal adhesion kinase (p‐FAK) (Y397) and total FAK in Talin1‐silenced SK‐Hep‐1 and HepG2 cells was determined using Western blot analysis. p‐FAK (Y397) was quantified as integrated density to represent the amount of protein. Numerical values represent the quantified data as normalized to the scramble group. (b) Western blot analysis for the expression of p‐ERK1/2, total ERK1/2, p‐Smad2/3, total Smad2/3, phosphorylated protein kinase B (p‐AKT), total AKT, RhoA, and Rac1 in Talin1‐silenced HCC cells. (c). Western blot analysis for the expression of p‐ERK1/2 and total ERK1/2 in SK‐Hep‐1 cells transfected with scrambled or Talin1‐specific siRNA#2 or #3. (d,e) Western blot analysis detected the expression of p‐ERK1/2, total ERK1/2, E‐cadherin, N‐cadherin, vimentin, and β‐catenin in SK‐Hep‐1 and HepG2 HCC cells transfected with scrambled or Talin1‐specific siRNA#1 and then treated with or without U0126 (10 μM) for 48 h. (f,g) Phase contrast microscopy detected the morphology of SK‐Hep‐1 cells and HepG2 cells transfected with scrambled or Talin1‐specific siRNA and then treated with or without U0126 (10 μM) for 48 h.
Figure 6
Figure 6
The ERK1/2 pathway is involved in Talin1 knockdown‐induced migration and invasion effects in hepatocellular carcinoma cells. (a,b) Transwell migration and matrix‐coated Transwell invasion assays were carried out to evaluate the migratory and invasive abilities of SK‐Hep‐1 cells transfected with scrambled or Talin1‐specific siRNA#1 or #2 and then treated with or without U0126 (10 μM) for 48 h. Top panels, representative images of Transwell migration (a) and matrix‐coated Transwell invasion (b) assays from indicated groups. Bottom panels, bar charts representing the average number of migratory (a) and invaded (b) cells from five random microscopic fields. **P < 0.01; ***P < 0.001. (c,d) Transwell migration and matrix‐coated Transwell invasion assays were carried out to evaluate the migratory and invasive abilities of HepG2 cells transfected with scrambled or Talin1‐specific siRNA#1 and then treated with or without U0126 (10 μM) for 24 h. Left panels, representative images of Transwell migration (c) and matrix‐coated Transwell invasion (d) assays from indicated groups. Right panels, bar charts representing the average number of migratory (c) and invaded (d) cells from five random microscopic fields. ***P < 0.001.
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