Connective Tissue Growth Factor Is Overexpressed In Human Hepatocellular Carcinoma And Promotes Cell Invasion And Growth

Objective: To determine the expression characteristics of connective tissuegrowth factor (CCN2) in human hepatocellular carcinoma (HCC) in histologyand to elucidate the roles of CCN2on hepatoma cell cycle progression andmetastasis in vitro.Methods: Liver samples from36patients (who underwent hepaticresection for the first HCC between2006and2011) and6normal individualswere examined for transforming growth factor β1(TGF-β1) or CCN2mRNA byin situ hybridization. Computer image analysis was performed to measureintegrated optimal density of CCN2mRNA-positive cells in carcinoma foci andthe surrounding stroma. Fibroblast-specific protein-1(FSP-1) and E-cadherinwere examined to evaluate the process of epithelial to mesenchymal transition,α-smooth muscle actin （α-SMA）and FSP-1were detected to identify hepaticstellate cells, and CD34was measured to evaluate the extent of vascularizationin liver tissues by immunohistochemical staining. CCN2was assessed for itsstimulation of HepG2cell migration and invasion using commercial kits whileflow cytometry was used to determine CCN2effects on HepG2cell-cycle. Results: In situ hybridization analysis showed that TGF-β1mRNA wasmainly detected in connective tissues and vasculature around carcinoma foci. Incomparison to normal controls, CCN2mRNA was enhanced1.9-fold incarcinoma foci (12.36±6.08vs6.42±2.35) or9.4-fold in the surroundingstroma (60.27±28.71vs6.42±2.35), with concomitant expression of CCN2and TGF-β1mRNA in those areas. Epithelial-mesenchymal transitionphenotype related with CCN2was detected in12/36(33.3%) of HCC liversamples at the edges between carcinoma foci and vasculature. Incubation ofHepG2cells with CCN2(100ng/mL) resulted in more of the cells transitioninginto S phase (23.85±2.35vs10.94±0.23), and induced a significant migratory(4.0-fold) and invasive (5.7-fold) effect. TGF-β1-induced cell invasion wasabrogated by a neutralizing CCN2antibody showing that CCN2is adownstream mediator of TGF-β1-induced hepatoma cell invasion.Conclusion： These data support a role for CCN2in the growth andmetastasis of HCC and highlight CCN2as a potential novel therapeutic target.