The role of hepatocyte nuclear factor 4alpha in metastatic tumor formation of hepatocellular carcinoma and its close relationship with the mesenchymal-epithelial transition markers

Abstract

Background: Mesenchymal-epithelial transition (MET) is now suggested to participate in the process of metastatic tumor formation. However, in hepatocellular carcinoma (HCC) the process is still not well revealed.

Methods: Paraffin-embedded tissue samples were obtained from 13 patients with HCC in Shengjing Hospital of China Medical University. The expression of E-cadherin, Fibronectin, N-cadherin, Vimentin, Hepatocyte nuclear factor 4alpha (HNF4alpha), Snail and Slug was assessed in primary tumors and their corresponding metastases by immunohistochemical staining. Next, the expression of HNF4alpha and E-cadherin in four HCC cell lines was examined. Furthermore, SK-Hep-1 cells were transfected with human HNF4alpha expression vector, and the change of E-cadherin expression was assessed.

Results: 45.2% (14/31) of the lesions in the metastases showed increased E-cadherin expression compared with the primaries, suggesting the possible occurrence of MET in metastatic tumor formation of HCC, as re-expression of E-cadherin is proposed to be the important hallmark of MET. The occurrence of MET was also confirmed by the reduced expression of Fibronectin (54.8%, 17/31), N-cadherin (38.7%, 12/31) and Vimentin (61.3%, 19/31) in the metastases. 45.2% (14/31) of the lesions in the metastases also showed increased HNF4alpha expression, and 67.7% (21/31) and 48.4% (15/31) of metastases showed decreased Snail and Slug expression respectively. Statistical results showed that the expression of HNF4alpha was positively related with that of E-cadherin, and negatively correlated with that of Snail, Slug and Fibronectin, suggesting that the expression change of the MET markers in the metastatic lesions might be associated with HNF4alpha. Among the four HCC cell lines, both HNF4alpha and E-cadherin expressed high in Hep3B and Huh-7 cells, but low in SK-Hep-1 and Bel-7402 cells. Furthermore, the expression of E-cadherin increased accordingly when SK-Hep-1 cells were transfected with human HNF4alpha expression vector, further confirming the role of HNF4alpha in the regulation of E-cadherin expression.

Conclusions: Our clinical observations and experimental data indicate that HNF4alpha might play a crucial role in the metastatic tumor formation of HCC, and the mechanism may be related with the process of phenotype transition.

Figure 1

The expression of MET marks in primary tumors and their corresponding metastases suggested the occurrence of MET in the metastases. A) The number of the lesions with different staining intensity of the MET marks (including E-cadherin, N-cadherin, Fibronectin and Vimentin) in the primary tumors and their corresponding metastases. B) The expression change of MET marks in the metastases compared with their corresponding primary tumors (increased E-cadherin, decreased N-cadherin, Fibronectin and Vimentin expression), marking via the lines, showed the occurrence of MET in the metastases. C) The examples of the cases with increased E-cadherin, decreased N-cadherin, Fibronectin and Vimentin expression in the metastases compared with their primary tumors were showed. In the normal tumor-adjacent tissue, the expression of E-cadherin was mainly strong, but the expression of N-cadherin, Fibronectin and Vimentin was usually negative.

Figure 2

The expression of HNF4alpha, slug and snail in primary tumors and their corresponding metastases. A) The number of the lesions with different staining intensity of HNF4alpha, Slug and Snail in the primary tumors and its metastases. B) The expression change of HNF4alpha, Slug and Snail in the metastases compared with their corresponding primary tumors, marking via the lines, showed the role of HNF4alpha, Slug and Snail in the metastases. C) The examples of the cases with increased HNF4alpha, decreased Slug and Snail expression in the metastases compared with their primary tumors were showed. In the normal tumor-adjacent tissue, the expression of HNF4alpha was mainly strong, while the expression of Slug and Snail was usually negative.

Figure 3

Correlation of the E-cadherin and HNF4alpha expression showed in four HCC cell lines. The expression of E-cadherin and HNF4alpha was detected in four HCC cell lines by western blot examination. It was showed that E-cadherin and HNF4alpha protein expression were both strong in Hep3B and Huh-7 cells, but weak in SK-hep-1 and BEL-7402 cells, showing the correlation of the E-cadherin and HNF4alpha expression.

Figure 4

The expression of HNF4alpha, E-cadherin and vimentin in SK-hep-1 and Hep3B cells. Immunofluorescence examination showed again the expression of E-cadherin and HNF4alpha in Hep3B cells was stronger than that in SK-hep-1 cells. In addition, it was found that the expression of vimentin in SK-hep-1 cells is similar with that in Hep3B cells.

Figure 5

HNF4alpha regulated the expression of E-cadherin in HCC cells. The SK-Hep-1 cells were transfected with HNF4alpha-expression plasmid. As the expression of HNF4alpha increased, the expression of E-cadherin correspondingly increased. A) Transient transfection. B) Stable transfection.