Véronique Neaud

Myofibroblasts are responsible for collagen synthesis in the stroma of human hepatocellular carcinoma: an in vivo and in vitro study

BACKGROUND/AIMS Marked changes in extracellular matrix occur in the stroma of hepatocellular carcinoma, as compared to normal or cirrhotic liver. The cell types responsible for extracellular matrix synthesis within hepatocellular carcinoma have not been clearly identified. METHODS In vivo collagen synthesis was studied by in situ hybridization and immunohistochemistry for types I, IV, V and VI collagen, together with immunolabeling of alpha-smooth muscle actin, a myofibroblast marker, and CD34, an endothelial cell marker. In vitro, extracellular matrix deposition by cultured myofibroblasts was studied by reticulin staining, immunocytochemistry and RNase protection. RESULTS All collagens studied were expressed in the stroma of the tumor, with a higher level of type VI and IV collagens than of type I and V. The majority of the cells expressing collagen transcripts in human hepatocellular carcinoma stroma were alpha-actin positive and CD 34 negative. In vitro experiments demonstrated that the hepatocellular carcinoma cell lines HepG2, HuH7 and Hep3B markedly increased extracellular matrix deposition by human liver myofibroblasts. This increase was mediated by a soluble mediator present in tumor cell conditioned medium. It was not explained by an increase in mRNA levels of extracellular matrix components, nor by a decrease in the secretion of matrix-degrading proteinases by myofibroblasts. CONCLUSIONS Myofibroblasts are the main source of collagens in the stroma of hepatocellular carcinoma. Our data also indicate that tumoral hepatocytes increase extracellular matrix deposition by cultured myofibroblasts, probably by post-transcriptional mechanisms. The generation of hepatocellular carcinoma stroma by myofibroblasts could thus be under control of tumoral cells.

Direct evidence that hepatocyte growth factor-induced invasion of hepatocellular carcinoma cells is mediated by urokinase

BACKGROUND/AIMS We have shown that hepatocyte growth factor secreted by human hepatic myofibroblasts increased the in vitro invasion of the hepatocarcinoma cell line HepG2 through Matrigel. Our aim in this study was to evaluate the role of urokinase in this process. METHODS Expression of urokinase in HepG2 cells was measured by Northern blot and zymography, and plasminogen activation was shown by a chromogenic substrate assay. Cell invasion was assayed on Matrigel-coated filters. Urokinase and urokinase receptor transcripts in hepatocarcinoma were detected by reverse transcription-polymerase chain reaction. Activated hepatocyte growth factor was detected by Western blot with a hepatocyte growth factor-beta chain-specific antibody. RESULTS HepG2 cells expressed urokinase mRNA and secreted active urokinase. Urokinase expression was enhanced by hepatocyte growth factor at the protein and mRNA level. Notably, cell-surface-associated urokinase was increased 22-fold by hepatocyte growth factor. Hepatocyte growth factor also increased urokinase receptor mRNA expression. B428, a urokinase inhibitor, decreased by up to 70% HepG2 invasion induced by myofibroblasts and by 90% that induced by recombinant hepatocyte growth factor. This was not due to a decrease in the generation of activated hepatocyte growth factor by myofibroblasts. Finally, all 17 hepatocarcinoma samples tested expressed urokinase and urokinase receptor transcripts. CONCLUSION Hepatocyte growth factor-dependent, myofibroblasts-induced invasion of HepG2 cells is secondary to the induction of urokinase expression on tumor cells.

Expression of tissue factor pathway inhibitor-2 in murine and human liver regulation during inflammation

Tissue factor pathway inhibitor-2 (TFPI-2) is a recently described serine proteinase inhibitor. Human and murine TFPI-2 share about 50% homology. The aim of this study was to investigate the cellular localization of human and murine TFPI-2 in the liver and the regulation of their expression during acute inflammation. Northern blot, in situ hybridization and studies on isolated hepatocytes demonstrated a high-level expression of TFPI-2 in murine hepatocytes. On the other hand, very little TFPI-2 mRNA expression could be detected in human liver. Studies with isolated human liver cells suggested that TFPI-2 expression in human liver was mainly observed in liver sinusoidal endothelial cells rather than hepatocytes. Liver murine TFPI-2 expression was greatly increased after lipopolysaccharide administration with a delayed kinetics as compared to alpha1-acid glycoprotein, a classical acute-phase reactant. Accordingly, studies with isolated cells showed that the increase in TFPI-2 transcripts occurred in non-hepatocytic cells. Moreover, the LPS response was abolished in mice with a hepatocyte-specific KO for the gp130 receptor, thus indicating that a mediator from hepatocytes is involved in the up-regulation of TFPI-2 in non-parenchymal cells. In conclusion, murine TFPI-2 is highly expressed in hepatocytes in the normal murine liver and is upregulated in non-parenchymal cells in the context of inflammation. The large difference in the level of liver expression of human and murine TFPI-2 suggests that despite significant sequence similarities, these proteins presumably have different functions in the two species.

Reptin Regulates DNA Double Strand Breaks Repair in Human Hepatocellular Carcinoma

Reptin/RUVBL2 is overexpressed in most hepatocellular carcinomas and is required for the growth and viability of HCC cells. Reptin is involved in several chromatin remodeling complexes, some of which are involved in the detection and repair of DNA damage, but data on Reptin involvement in the repair of DNA damage are scarce and contradictory. Our objective was to study the effects of Reptin silencing on the repair of DNA double-strand breaks (DSB) in HCC cells. Treatment of HuH7 cells with etoposide (25 μM, 30 min) or γ irradiation (4 Gy) increased the phosphorylation of H2AX by 1.94 ± 0.13 and 2.0 ± 0.02 fold, respectively. These values were significantly reduced by 35 and 65 % after Reptin silencing with inducible shRNA. Irradiation increased the number of BRCA1 (3-fold) and 53BP1 foci (7.5 fold). Depletion of Reptin reduced these values by 62 and 48%, respectively. These defects in activation and/or recruitment of repair proteins were not due to a decreased number of DSBs as measured by the COMET assay. All these results were confirmed in the Hep3B cell line. Protein expression of ATM and DNA-PKcs, the major H2AX kinases, was significantly reduced by 52 and 61 % after Reptin depletion whereas their mRNA level remained unchanged. Phosphorylation of Chk2, another ATM target, was not significantly altered. Using co-immunoprecipitation, we showed an interaction between Reptin and DNA-PKcs. The half-life of newly-synthesized DNA-PKcs was reduced when Reptin was silenced. Finally, depletion of Reptin was synergistic with etoposide or γ irradiation to reduce cell growth and colony formation. In conclusion, Reptin is an important cofactor for the repair of DSBs. Our data, combined with those of the literature suggests that it operates at least in part by regulating the expression of DNA-PKcs by a stabilization mechanism. Overexpression of Reptin in HCC could be a factor of resistance to treatment, consistent with the observed overexpression of Reptin in subgroups of chemo-resistant breast and ovarian cancers.

Metalloproteinase meprin α regulates migration and invasion of human hepatocarcinoma cells and is a mediator of the oncoprotein Reptin

Hepatocellular carcinoma is associated with a high rate of intra-hepatic invasion that carries a poor prognosis. Meprin alpha (Mep1A) is a secreted metalloproteinase with many substrates relevant to cancer invasion. We found that Mep1A was a target of Reptin, a protein that is oncogenic in HCC. We studied Mep1A regulation by Reptin, its role in HCC, and whether it mediates Reptin oncogenic effects. MepA and Reptin expression was measured in human HCC by qRT-PCR and in cultured cells by PCR, western blot and enzymatic activity measurements. Cell growth was assessed by counting and MTS assay. Cell migration was measured in Boyden chambers and wound healing assays, and cell invasion in Boyden chambers. Silencing Reptin decreased Mep1A expression and activity, without affecting meprin β. Mep1A, but not meprin β, was overexpressed in a series of 242 human HCC (2.04 fold, p < 0.0001), and a high expression correlated with a poor prognosis. Mep1A and Reptin expressions were positively correlated (r = 0.39, p < 0.0001). Silencing Mep1A had little effect on cell proliferation, but decreased cell migration and invasion of HuH7 and Hep3B cells. Conversely, overexpression of Mep1A or addition of recombinant Mep1A increased migration and invasion. Finally, overexpression of Mep1A restored a normal cell migration in cells where Reptin was depleted. Mep1A is overexpressed in most HCC and induces HCC cell migration and invasion. Mep1A expression is regulated by Reptin, and Mep1A mediates Reptin-induced migration. Overall, we suggest that Mep1A may be a useful target in HCC.

Reptin regulates insulin-stimulated Akt phosphorylation in hepatocellular carcinoma via the regulation of SHP-1/PTPN6

Hepatocellular carcinoma (HCC) is the main primary cancer of the liver. Many studies have shown that insulin resistance is a risk factor for HCC. We previously discovered the overexpression and oncogenic role of the Reptin/RUVBL2 ATPase in HCC. Here, we found that Reptin silencing enhanced insulin sensitivity in 2 HCC cell lines, as shown by a large potentiation of insulin‐induced AKT phosphorylation on Ser473 and Thr308, and of downstream signalling. Reptin silencing did not affect the tyrosine phosphorylation of the insulin receptor nor of IRS1, but it enhanced the tyrosine phosphorylation of the p85 subunit of PI3K. The expression of the SHP‐1/PTPN6 phosphatase, which dephosphorylates p85, was reduced after Reptin depletion. Forced expression of SHP‐1 restored a normal AKT phosphorylation after insulin treatment in cells where Reptin was silenced, demonstrating that the downregulation of SHP1 is mechanistically linked to increased Akt phosphorylation. In conclusion, we have uncovered a new function for Reptin in regulating insulin signalling in HCC cells via the regulation of SHP‐1 expression. We suggest that the regulation of insulin sensitivity by Reptin contributes to its oncogenic action in the liver.