A Mouse Model of Hepatocellular Carcinoma (original) (raw)
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The American Journal of Pathology, 2002
Most mouse models of hepatocellular carcinoma have expressed growth factors and oncogenes under the control of a liver-specific promoter. In contrast, we describe here the formation of liver tumors in transgenic mice overexpressing human fibroblast growth factor 19 (FGF19) in skeletal muscle. FGF19 transgenic mice had elevated hepatic α-fetoprotein mRNA as early as 2 months of age, and hepatocellular carcinomas were evident by 10 months of age. Increased proliferation of pericentral hepatocytes was demonstrated by 5-bromo-2′-deoxyuridine incorporation in the FGF19 transgenic mice before tumor formation and in nontransgenic mice injected with recombinant FGF19 protein. Areas of small cell dysplasia were initially evident pericentrally, and dysplastic/neoplastic foci throughout the hepatic lobule were glutamine synthetase-positive, suggestive of a pericentral origin. Consistent with chronic activation of the Wingless/Wnt pathway, 44% of the hepatocellular tumors from FGF19 transgenic mice had nuclear staining for β-catenin. Sequencing of the tumor DNA encoding β-catenin revealed point mutations that resulted in amino acid substitutions. These findings suggest a previously unknown role for FGF19 in hepatocellular carcinomas.
Oncogene, 2008
Although fibroblast growth factor 19 (FGF19) can promote liver carcinogenesis in mice its involvement in human cancer is not well characterized. Here we report that FGF19 and its cognate receptor FGF receptor 4 (FGFR4) are coexpressed in primary human liver, lung and colon tumors and in a subset of human colon cancer cell lines. To test the importance of FGF19 for tumor growth, we developed an anti-FGF19 monoclonal antibody that selectively blocks the interaction of FGF19 with FGFR4. This antibody abolished FGF19-mediated activity in vitro and inhibited growth of colon tumor xenografts in vivo and effectively prevented hepatocellular carcinomas in FGF19 transgenic mice. The efficacy of the antibody in these models was linked to inhibition of FGF19-dependent activation of FGFR4, FRS2, ERK and b-catenin. These findings suggest that the inactivation of FGF19 could be beneficial for the treatment of colon cancer, liver cancer and other malignancies involving interaction of FGF19 and FGFR4.
Targeting FGFR4 Inhibits Hepatocellular Carcinoma in Preclinical Mouse Models
PLoS ONE, 2012
The fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling system plays critical roles in a variety of normal developmental and physiological processes. It is also well documented that dysregulation of FGF-FGFR signaling may have important roles in tumor development and progression. The FGFR4-FGF19 signaling axis has been implicated in the development of hepatocellular carcinomas (HCCs) in mice, and potentially in humans. In this study, we demonstrate that FGFR4 is required for hepatocarcinogenesis; the progeny of FGF19 transgenic mice, which have previously been shown to develop HCCs, bred with FGFR4 knockout mice fail to develop liver tumors. To further test the importance of FGFR4 in HCC, we developed a blocking anti-FGFR4 monoclonal antibody (LD1). LD1 inhibited: 1) FGF1 and FGF19 binding to FGFR4, 2) FGFR4-mediated signaling, colony formation, and proliferation in vitro, and 3) tumor growth in a preclinical model of liver cancer in vivo. Finally, we show that FGFR4 expression is elevated in several types of cancer, including liver cancer, as compared to normal tissues. These findings suggest a modulatory role for FGFR4 in the development and progression of hepatocellular carcinoma and that FGFR4 may be an important and novel therapeutic target in treating this disease.
Hepatology, 1996
It On six human hepatocellular carcinoma (HCC) cell affects vascular endothelial cells and stimulates angiogenelines (KIM-1, KYN-1, KYN-2, KYN-3, HAK-1A, and HAKsis, and it influences bFGF-responsive cells to change their 1B), we examined expressions and functions of the promorphology and growth pattern, and to increase their migrateins and messenger RNAs (mRNAs) of basic fibroblast tory activities. 11,16,18-20 To date, bFGF has been identified in growth factor (bFGF) and its receptor, i.e., fibroblast epithelial cells, endothelial cells, fibroblasts, macrophages, growth factor receptor-1 (FGFR-1), as well as mRNA exand extracellular matrix, in various organs in vivo, 11-14 and pressions of FGFR-2 Ç4. All six cell lines expressed the its presence has also been confirmed in tumor tissues. 11,12,21-23 proteins and mRNAs of bFGF and FGFR-1, and at least Schlze-Osthoff et al. 12 reported that tumors revealed a very one of FGFR-2 Ç4 mRNAs. Two of the six cell lines (KYNheterogeneous staining pattern, e.g., bFGF can be expressed 1 and KYN-3) presented significant release of bFGF in (1) only in tumor cells, (2) only on vascular endothelial cells, culture supernatant, while the release in the remaining or (3) only on macrophages. Identification of bFGF in tumor four cell lines was quite small. Addition of anti-bFGF tissues and in some cancerous cell lines allowed researchers neutralizing antibody (1, 10, or 20 mg/mL) to culture meto presume that bFGF is involved in the development and dium resulted in marked suppression of cell proliferaprogression of tumors. It is possible that bFGF produced by tion in all cell lines except HAK-1A. On the other hand, tumor cells affects proliferation of the tumor cells themselves addition of exogenous bFGF (0.1, 1, or 5 ng/mL) to culture through an autocrine or intracrine mechanism; or, bFGF acts medium stimulated cell proliferation except in KIM-1 on endothelial cells through a paracrine mechanism and and KYN-2. When KIM-1 was transplanted to nude mice stimulates angiogenesis; or bFGF induces higher productions and anti-bFGF antibody was injected subcutaneously to of plasminogen activators, various proteases, and collagena space surrounding the developed tumor, tumor prolifase, and contributes to infiltration and metastasis of tumor eration was significantly suppressed in nude mice that cells. 11,16,19,22 As high-affinity cell surface receptors for the received anti-bFGF antibody than in control mice, but FGF family, five types (fibroblast growth factor receptor there were no histological differences between the [FGFR]-1 Ç5) have been identified so far, 24-27 and their exgroups, including blood space formation in the stroma. pressions have been reported in various tumor tissues and In conclusion, hepatocellular carcinoma (HCC) cells cell lines, and in normal endothelial cells. 14,17,28-30 may possess a proliferation mechanism regulated by an Expression of bFGF in normal liver tissues has been a autocrine mechanism, a paracrine mechanism, or both, matter of controversy. Hughes and Hall 14 conducted an imwhich are mediated by bFGF/FGFR. (HEPATOLOGY munohistochemical study and observed bFGF expressions at 1996;24:198-205.) high levels in normal hepatocytes. However, other studies before them reported that normal hepatocytes did not express The fibroblast growth factor (FGF) family is a group of bFGF, or they only weakly expressed bFGF. 12,13 On the other structurally related multifunctional mitogenic polypeptides, hand, Motoo et al. 21 identified high bFGF expressions in the and its members possess heparin-binding property. At prescytoplasm of hepatocellular carcinoma (HCC) cells of 16 of ent, nine members, from FGF-1 to FGF-9, have been identi-56 patients (29%), and another study reported the cultured fied. 1-9 The FGF family has been widely distributed in normal HCC cell line, SK Hep-1, can produce bFGF in vitro. 31 Normal and/or tumor tissues, and they are known to take various hepatocytes do not express the bFGF receptor, FGFR-1, while important roles, e.g., in angiogenesis, tissue regeneration, a hepatoblastoma cell line (HepG2) has been reported to exwound healing, and embryonic development. 10-17 Among the press this receptor. 14,32 Consequently, expressions of bFGF/ FGF family, basic fibroblast growth factor (bFGF, or FGF-2) FGFR could be involved in the angiogenesis and proliferation acts as a potent mitogen and as a differentiation factor for of HCC cells, even though their precise mechanism has not various mesoderm-and neuroectoderm-derived cells. 11 bFGF been fully elucidated. In the present study, we examined (1) protein and messenger RNA (mRNA) expressions of bFGF and FGFR1 Ç4, and (2) proliferation effects of bFGF on HCC cells through an Abbreviations: FGF, fibroblast growth factor; bFGF, basic fibroblast growth factor; autocrine mechanism both in vitro and in nude mice.
1998
The fibroblast growth factors (FGFs) play key roles in controlling tissue growth, morphogenesis, and repair in animals. We have cloned a novel member of the FGF family, designated FGF-18, that is expressed primarily in the lungs and kidneys and at lower levels in the heart, testes, spleen, skeletal muscle, and brain. Sequence comparison indicates that FGF-18 is highly conserved between humans and mice and is most homologous to FGF-8 among the FGF family members. FGF-18 has a typical signal sequence and was glycosylated and secreted when it was transfected into 293-EBNA cells. Recombinant murine FGF-18 protein (rMuFGF-18) stimulated proliferation in the fibroblast cell line NIH 3T3 in vitro in a heparan sulfate-dependent manner. To examine its biological activity in vivo, rMuFGF-18 was injected into normal mice and ectopically overexpressed in transgenic mice by using a liver-specific promoter. Injection of rMuFGF-18 induced proliferation in a wide variety of tissues, including tissues of both epithelial and mesenchymal origin. The two tissues which appeared to be the primary targets of FGF-18 were the liver and small intestine, both of which exhibited histologic evidence of proliferation and showed significant gains in organ weight following 7 (sometimes 3) days of FGF-18 treatment. Transgenic mice that overexpressed FGF-18 in the liver also exhibited an increase in liver weight and hepatocellular proliferation. These results suggest that FGF-18 is a pleiotropic growth factor that stimulates proliferation in a number of tissues, most notably the liver and small intestine.
Proceedings of the National Academy of Sciences, 1997
Hepatocyte growth factor͞scatter factor (HGF͞SF) is a mesenchymally derived, multifunctional paracrine regulator possessing mitogenic, motogenic, and morphogenetic activities in cultured epithelial cells containing its tyrosine kinase receptor, Met. c-met has been implicated in oncogenesis through correlation of expression with malignant phenotype in specific cell lines and tumors. Paradoxically, however, HGF͞SF can also inhibit the growth of some tumor cells. To elucidate the oncogenic role of HGF͞SF in vivo, transgenic mice were created such that HGF͞SF was inappropriately targeted to a variety of tissues. HGF͞SF transgenic mice developed a remarkably broad array of histologically distinct tumors of both mesenchymal and epithelial origin. Many neoplasms arose from tissues exhibiting abnormal development, including the mammary gland, skeletal muscle, and melanocytes, suggesting a functional link between mechanisms regulating morphogenesis and those promoting tumorigenesis. Most neoplasms, especially melanomas, demonstrated overexpression of both the HGF͞SF transgene and endogenous c-met, and had enhanced Met kinase activity, strongly suggesting that autocrine signaling broadly promotes tumorigenesis. Thus, subversion of normal mesenchymalepithelial paracrine regulation through the forced misdirection of HGF͞SF expression induces aberrant morphogenesis and subsequent malignant transformation of cells of diverse origin.
Hepatology, 2011
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 per-sistent 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; siSCR, scrambled small interfering RNA; Tris, trishydroxymethylaminomethane; vEGF, vascular endothelial growth factor.
Oncogene, 1999
Hepatocyte growth factor (HGF) is a polypeptide with mitogenic, motogenic, and morphogenic eects on dierent cell types including hepatocytes. HGF is expressed as two biologically active isotypes resulting from alternative RNA splicing. The roles of each HGF isoform in development, liver regeneration and tumorigenesis have not yet been well characterized. We report the generation and analysis of transgenic mice overexpressing the ®ve amino acid-deleted variant of HGF (dHGF) in the liver by virtue of an albumin expression vector. These ALB-dHGF transgenic mice develop normally, have an enhanced rate of liver regeneration after partial hepatectomy, and exhibit a threefold higher incidence of hepatocellular carcinoma (HCC) beyond 17 months of age. Moreover, overexpression of dHGF dramatically accelerates diethyl-nitrosamine induced HCC tumorigenesis. These tumors arise faster, are signi®cantly larger, more numerous and more invasive than those appearing in non-transgenic littermates. Approximately 90% of female dHGF-transgenic mice had multiple macroscopic HCCs 40 weeks after injection of DEN; whereas the non-transgenic counterparts had only microscopic nodules. Liver tumors and cultured tumor cell lines from dHGF transgenics showed high levels of HGF and c-Met mRNA and protein. Together, these results reveal that in vivo dHGF plays an active role in liver regeneration and HCC tumorigenesis.
Proceedings of the National Academy of Sciences, 1996
Overexpression of the c-myc oncogene is associated with a variety of both human and experimental tumors, and cooperation of other oncogenes and growth factors with the myc family are critical in the evolution of the malignant phenotype. The interaction of hepatocyte growth factor (HGF) with c-myc during hepatocarcinogenesis in a transgenic mouse model has been analyzed. While sustained overexpression of c-myc in the liver leads to cancer, coexpression of HGF and c-myc in the liver delayed the appearance of preneoplastic lesions and prevented malignant conversion. Furthermore, tumor promotion by phenobarbital was completely inhibited in the c-myc/HGF double transgenic mice, whereas phenobarbital was an effective tumor promoter in the c-myc single transgenic mice. The results indicate that HGF may function as a tumor suppressor during early stages of liver carcinogenesis, and suggest the possibility of therapeutic application for this cytokine.