Expression of hepatocyte growth factor in human hepatocellular carcinoma.

BACKGROUND/AIMS: We have shown that hepatocyte growth factor, secreted by human liver myofibroblasts, promoted in vitro invasion of human hepatocellular carcinoma cell lines. The aim of this work was to measure hepatocyte growth factor expression in 29 human hepatocellular carcinomas and the corresponding peri-tumoral livers. METHODS: We used reverse transcription-polymerase chain reaction, in situ hybridization, ELISA and Western blot. RESULTS: Sixty-two of tested hepatocellular carcinomas were positive by reverse transcription-polymerase chain reaction. With in situ hybridization, a signal was found in every sample. In many cases, the signal was localized in cells labeled with an anti-smooth muscle alpka-actin antibody, while hepatocytes were mostly non-labeled. ELISA, performed in 15 pairs of hepatocellular carcinomas and surrounding livers, detected hepatocyte growth factor in every sample with wide variations. Hepatocellular carcinomas that had developed in non-cirrhotic livers contained essentially the same amount of hepatocyte growth factor as the matching non-tumoral liver. In cirrhotic livers, the hepatocyte growth factor content of the tumors was significantly lower than that of the surrounding cirrhotic livers. CONCLUSIONS: These data indicate that hepatocyte growth factor is expressed at significant levels in every hepatocellular carcinoma tested and that its expression takes place in the stromal myofibroblasts.

Hepatocarcinoma cells stimulate hepatocyte growth factor secretion in human liver myofibroblasts.

Hepatocellular carcinoma (HCC), the main type of primary liver cancer, is characterized by a high rate of intra-hepatic invasion. The stroma of HCC is infiltrated by myofibroblasts. We have previously shown that hepatocyte growth factor (HGF) secreted by human liver myofibroblasts greatly increased the in vitro invasiveness of 3 human HCC cell lines. In this study we show that the conditioned medium (CM) from the same HCC cell lines dose-dependently stimulates HGF secretion by myofibroblasts. This effect was post-transcriptional as no increase in HGF mRNA was observed. We show that the effect of CM is not due to IL-1, IL-6, IGF-1, bFGF or PDGF, previously shown to stimulate HGF synthesis in other models. Our data demonstrate that HCC cells increase HGF secretion by liver myofibroblasts in a paracrine way that could act to enhance invasion.

Osteonectin/SPARC is overexpressed in human hepatocellular carcinoma.

Osteonectin (ON)/SPARC is a glycoprotein involved in extracellular matrix remodelling. ON expression by myofibroblasts has been reported in fibrotic human liver. As ON also plays a role in cell adhesion, differentiation, and proliferation, this study was designed to document its expression in human hepatocellular carcinoma (HCC). Tissues from 26 HCCs of various histological grades and architecture and from surrounding non-tumour liver (23 cirrhotic or fibrotic, three non-fibrotic) were tested by in situ hybridization and immunohistochemistry. Immunohistochemical detection of alpha-smooth muscle actin (alpha-SMA) was performed on serial sections or in combination with hybridization. Large amounts of ON mRNA and protein were detected in the tumour capsule, in the fibrous bands, and along capillaries within HCCs. The signal was located in cells suggestive of myofibroblasts, as confirmed by positive staining for alpha-SMA. In HCC, ON protein was always detectable, with strong staining in high-grade tumours, whereas it was mostly undetectable in non-tumour tissues. A clear difference was also shown for ON transcripts, except in a few cases with chronic active hepatitis, where ON transcripts were also expressed at a high level. Overexpression of ON transcripts in HCC vs. non-tumour liver was confirmed by RNA blot in 20/22 patients tested. In conclusion, ON is strongly expressed by the stromal myofibroblasts of human HCC, especially of high grade. This expression could play a role in tumour progression.

Human hepatic myofibroblasts increase invasiveness of hepatocellular carcinoma cells: evidence for a role of hepatocyte growth factor.

The stroma of hepatocellular carcinomas (HCC) is infiltrated with myofibroblasts (MFs). Preliminary in vivo data have suggested that liver MF express hepatocyte growth factor (HGF), a cytokine that has been implicated in several tumor models. Our aim was to investigate the role of MF and HGF in HCC. Cultured liver MF expressed HGF messenger RNA (mRNA) and secreted HGF in their medium, as shown by Western blot, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Addition of MF-conditioned medium to the HepG2 HCC cell line induced cell scattering. This was associated with a decrease in cell proliferation. MF also increased about 100-fold the ability of HepG2 to invade Matrigel. Increased invasiveness was also shown for HuH7 cells, but no scattering was observed and cell proliferation was stimulated. All the effects of MF on both tumor cell types were blocked by addition of an antibody to HGF and they all could be reproduced by adding recombinant HGF to the tumor cells. RT-PCR and Western blot analysis confirmed that both tumor cell lines expressed c-met, the receptor for HGF. The effects of MF-conditioned medium were not reproduced by acidic fibroblast growth factor, basic fibroblast growth factor, epidermal growth factor (EGF), transforming growth factor-beta1 (TGF-beta1), or platelet-derived growth factor (PDGF-BB). Reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that HGF was expressed in human HCC. Our data show that human liver MF act on HCC cells to increase their invasiveness and suggest that MF-derived HGF could be involved in the pathogenesis of HCC.