title: Connective tissue growth factor is overexpressed in human hepatocellular carcinoma and promotes cell invasion and growth.
abstract: OBJECTIVE
  To determine the expression characteristics of connective tissue growth factor (CTGF/CCN2) in human hepatocellular carcinoma (HCC) in histology and to elucidate the roles of CCN2 on hepatoma cell cycle progression and metastasis in vitro.
  
  
  METHODS
  Liver samples from 36 patients (who underwent hepatic resection for the first HCC between 2006 and 2011) and 6 normal individuals were examined for transforming growth factor β1 (TGF-β1) or CCN2 mRNA by in situ hybridization. Computer image analysis was performed to measure integrated optimal density of CCN2 mRNA-positive cells in carcinoma foci and the surrounding stroma. Fibroblast-specific protein-1 (FSP-1) and E-cadherin were examined to evaluate the process of epithelial to mesenchymal transition, α-smooth muscle actin and FSP-1 were detected to identify hepatic stellate cells, and CD34 was measured to evaluate the extent of vascularization in liver tissues by immunohistochemical staining. CCN2 was assessed for its stimulation of HepG2 cell migration and invasion using commercial kits while flow cytometry was used to determine CCN2 effects on HepG2 cell-cycle.
  
  
  RESULTS
  In situ hybridization analysis showed that TGF-β1 mRNA was mainly detected in connective tissues and vasculature around carcinoma foci. In comparison to normal controls, CCN2 mRNA was enhanced 1.9-fold in carcinoma foci (12.36 ± 6.08 vs 6.42 ± 2.35) or 9.4-fold in the surrounding stroma (60.27 ± 28.71 vs 6.42 ± 2.35), with concomitant expression of CCN2 and TGF-β1 mRNA in those areas. Epithelial-mesenchymal transition phenotype related with CCN2 was detected in 12/36 (33.3%) of HCC liver samples at the edges between carcinoma foci and vasculature. Incubation of HepG2 cells with CCN2 (100 ng/mL) resulted in more of the cells transitioning into S phase (23.85 ± 2.35 vs 10.94 ± 0.23), and induced a significant migratory (4.0-fold) and invasive (5.7-fold) effect. TGF-β1-induced cell invasion was abrogated by a neutralizing CCN2 antibody showing that CCN2 is a downstream mediator of TGF-β1-induced hepatoma cell invasion.
  
  
  CONCLUSIONS
  These data support a role for CCN2 in the growth and metastasis of HCC and highlight CCN2 as a potential novel therapeutic target.
authors: Department of Hepatic-Biliary-Pancreatic Medicine, First Hospital, Jilin University, Changchun 130021, Jilin Province, China.
authors: Ming
authors: M
authors: Xiu
authors: Ya-Hui
authors: YH
authors: Liu
authors: David R
authors: DR
authors: Brigstock
authors: Fang-Hui
authors: FH
authors: He
authors: Rui-Juan
authors: RJ
authors: Zhang
authors: Run-Ping
authors: RP
authors: Gao
mesh_terms: D000328:Adult; D000368:Aged; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D049109:Cell Proliferation; D055513:Connective Tissue Growth Factor / Q000378:metabolism*; D018450:Disease Progression; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D056945:Hep G2 Cells; D006801:Humans; D007150:Immunohistochemistry; D008099:Liver / Q000473:pathology; D008113:Liver Neoplasms / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: C526317:CCN2 protein, human; D012333:RNA, Messenger; D055513:Connective Tissue Growth Factor
keywords: Connective tissue growth factor; Hepatocellular carcinoma; Hepatoma cell line; Invasion; Migration