Fibroblast growth factors (FGFs) and their high-affinity receptors [fibroblast growth factor receptors (FGFRs)] contribute to autocrine and paracrine growth stimulation in several nonliver cancer entities. Here we report that at least one member of the FGF8 subfamily (FGF8, FGF17, and FGF18) was up-regulated in 59% of 34 human hepatocellular carcinoma (HCC) samples that we investigated. The levels of the corresponding receptors (FGFR2, FGFR3, and FGFR4) were also elevated in the great majority of the HCC cases. Overall, 82% of the HCC cases showed overexpression of at least one FGF and/or FGFR. The functional implications of the deregulated FGF/FGFR system were investigated by the simulation of an insufficient blood supply. When HCC-1.2, HepG2, or Hep3B cells were subjected to serum withdrawal or the hypoxia-mimetic drug deferoxamine mesylate, the expression of FGF8 subfamily members increased dramatically. In the serum-starved cells, the incidence of apoptosis was elevated, whereas the addition of FGF8, FGF17, or FGF18 impaired apoptosis, which was associated with phosphorylation of extracellular signal-regulated kinase 1/2 and ribosomal protein S6. In contrast, down-modulation of FGF18 by small interfering RNA (siRNA) significantly reduced the viability of the hepatocarcinoma cells. siRNA targeting FGF18 also impaired the cells' potential to form clones at a low cell density or in soft agar. With respect to the tumor microenvironment, FGF17 and FGF18 stimulated the growth of HCC-derived myofibroblasts, and FGF8, FGF17, and FGF18 induced the proliferation and tube formation of hepatic endothelial cells. Conclusion: FGF8, FGF17, and FGF18 are involved in autocrine and paracrine signaling in HCC and enhance the survival of tumor cells under stress conditions, malignant behavior, and neoangiogenesis. Thus, the FGF8 subfamily supports the development and progression of hepatocellular malignancy. (HEPATOLOGY 2011;53:854-864) H epatocellular carcinoma (HCC) is the thirdleading cause of cancer deaths worldwide. 1 Important risk factors for this disease are persistent infections with hepatitis viruses and chronic steatohepatitis due to ethanol abuse and obesity, which contribute to the increasing incidence of HCC in Abbreviations: AHR, aryl hydrocarbon receptor; AKT, protein kinase B; ERK, extracellular signal-regulated kinase; ETS, E twenty-six; FACS, fluorescenceactivated cell sorting; FBS, fetal bovine serum; FCS, fetal colf serum; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; GSK3b, glycogen synthase kinase 3b; HCC, hepatocellular carcinoma; HIF, hypoxia inducible factor; MAP, mitogen-activated protein; MF, myofibroblast; mRNA, messenger RNA; MTF, metal-responsive transcription factor; pERK, phosphorylated extracellular signal-regulated kinase; pGSK3b, phosphorylated glycogen synthase kinase 3b; pS6, phosphorylated S6; qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; siFGF18, small interfering RNA targeting fibroblast growth factor 18; siRNA, small interfering RNA;...

Hepatocytes are known to express a large number of characteristic proteins. Transformed and cultured hepatocytes only partially maintain functional cell differentiation characteristics, which can be assessed by proteome profiling. Here, we applied 2D-PAGE analysis in addition to shotgun proteomics to assess the functional cell state of primary human hepatocytes (PHH), HepG2 and Hep3B cells. Out of a total of 1995 proteins identified in the cytoplasm of these cells, we filtered 107 proteins which are characteristic for hepatocytes. A total of 104 of those were identified in primary human hepatocytes, 20 in HepG2, and only 6 in Hep3B. Forty-six out of 72 proteins identified in the secretome of PHH, 55 out of 139 in HepG2, and only 24 out of 72 in Hep3B were plasma proteins characteristic for hepatocytes. Beside other biomarker candidates presently identified, 11 proteins of the HepG2 secretome have been described previously as biomarkers for hepatocellular carcinoma. Because of indications that epithelial to mesenchymal transition (EMT) may have occurred in the cultured hepatoma cells, we included the analysis of fibroblasts representative for mesenchymal cells. Hep3B, but not HepG2, secreted five proteins including follistatin-related protein 1 which are characteristic for mesenchymal cells and may be marker proteins for EMT. Our data demonstrate that HepG2 show more features characteristic for hepatocytes than Hep3B, while Hep3B express more mesenchymal proteins indicative for EMT. Proteome profiling thus proved to enable comprehensive assessment of functional cell states and cell differentiation states of cultured hepatocytes and enabled the identification of numerous biomarkers for hepatocellular carcinoma and EMT.

No abstract

No abstract