Expression of Connective Tissue Growth Factor is Involved in Tumor Cell Growth and Metastasis in Human Hepatocellular Carcinoma

Abstract

Purpose: Recent studies have shown that connective tissue growth factor (CTGF/CCN2) acts as a downstream mediator for TGF-β1-induced tumor growth in hepatocellular carcinoma (HCC) in animal models and in vitro studies. The aim of this study was to investigate the expressional characteristics of CCN2 in human HCC, the relation between CCN2 and epithelial-mesenchymal transation (EMT) process and the effects of CCN2 in hepatoma cell proliferation, migration and invasion. Methods: Liver samples from 36 patients (who underwent hepatic resection for the first HCC) and 6 normal individuals were used to examine TGF-β1 and CCN2 mRNA by in situ hybridization. The process of EMT, identification of hepatic stellate cells and distribution of vasculatures in the liver tissue sections were evaluated by immunohistochemistry staining. Computer image analysis was performed to measure the integrated optimal density (IOD) of CCN2 mRNA-positive cells in carcinoma foci and in surrounding stroma respectively. CCN2 was assessed for its stimulation of HepG2 cell proliferation, migration and invasion by using commercial kits. Results: In situ hybridization analysis showed a significant increase of TGF-β1 mRNA was detected mainly in connective tissues and vesculatures around carcinoma foci and that a 9.4-fold enhanced expression of CCN2 mRNA was seen in the surrounding stroma and only a 1.9-fold expression of CCN2 mRNA in carcinoma foci, comparing with normal controls (p<0.01). The results above exhibited a concomitant expression of CCN2 and TGF-β1 mRNA in the surrounding stroma. EMT phenotype related with CCN2 was detected in 12/36 (33.3%) of HCC liver samples at the edges between carcinoma foci and vesculatures. Incubation of HepG2 cells with CCN2 at a concentration of 100 ng/ml induced a significant proliferious (1.7-fold), migratory (4.0-fold) and invasive (5.7-fold) effect. Furthermore, TGF-β1-induced HepG2 cell invasion was abrogated in the presence of neutralizing CCN2 antibody, suggesting that CCN2 acts as a downstream mediator for TGF-β1-induced HepG2 cell invasion. Conclusion: These data reveal the role of CCN2 in the growth and metastasis of HCC and highlight CCN2 as potential novel therapeutic targets.