A Mouse Model of Hepatocellular Carcinoma: Ectopic Expression of Fibroblast Growth Factor 19 in Skeletal Muscle of Transgenic Mice (original) (raw)
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A Mouse Model of Hepatocellular Carcinoma
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 ␣-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 -catenin. Sequencing of the tumor DNA encoding -catenin revealed point mutations that resulted in amino acid substitutions. These findings suggest a previously unknown role for FGF19 in hepatocellular carcinomas.
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.
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.
Digestive Diseases and Sciences, 2015
Purpose In this study, we explored whether treatment with FGF-21 could prevent diethylnitrosamine (DEN) induced hepatocarcinogenesis in mice. Methods & Results Hepatoma was induced by injection of DEN every three days for 18 weeks. For the prophylactic experiment, mice were firstly injected with FGF-21 for 2 weeks, then FGF-21 was administered to the mice once daily in association with DEN injection till the end of the experiment. The hepatoma incidence of mice treated with FGF-21 was 13.3 %, while the incidence of mice treated with saline was 61.5 %. To understand the mechanisms, we compared the expression of bklotho (KLB) and oxidative stress level in the livers between the mice treated with FGF-21 and saline. We found that FGF-21 could suppress DEN-induced oxidative stress and up-regulate the expression of KLB in the livers. To confirm these results, we compared the expression of KLB in L02 cells stimulated with or without FGF-21. Besides, we established DENinduced oxidative stress cell model to affirm the relationship between FGF-21 and DEN-induced oxidative stress in vitro. Results showed that FGF-21 increased the expression of KLB and diminished the DEN-induced oxidative stress in vitro in a dose dependent manner. Conclusion Systemic administration of FGF-21 can prevent DEN-induced hepatocarcinogenesis via suppressing oxidative stress and increasing the expression of KLB.
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.
2008
AIM: The study was aimed to investigate the dynamic alteration of hepatic trans- forming growth factor (TGF)-β1 expression during the formation of hepatocellular carcinoma. METHODS: The animal model for hepatocarcinogenesis was made with male Spra- gue-Dawley (SD) rats induced with 0.05% of 2-fluorenylacetamide. Morphological changes in rat livers were examined by Hematoxylin and Eosin staining. Hepatic total RNA was extracted from rat livers and TGF-β1 mRNA level was analyzed by nested RT- PCR. The levels of TGF-β1 in liver and blood were quantitatively detected by ELISA. Simultaneously, hepatocyte distribution of TGF-β1 was analyzed by immunohisto- chemistry. RESULTS: Our histopathological data confirmed the 2-fluorenylacetamide-induced malignant transformation of rat hepatocytes progressed from granule-like degeneration to atypical hyperplasia to hepatoma formation. The expression levels of rat hepatic total RNA, TGF-β1 mRNA, and TGF-β1 protein were significantly increased with t...
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.
Cancer Research, 1994
Transforming growth factor a (TGF-a) is a polypeptide closely asso elated with hepatocyte proliferation in vivo and in vitro. In order to inves tigate the mechanisms by which TGF-a contributes to hepatocyte repli cation and transformation, we isolated hepatocytes from mice bearing a human TGF-a transgene and examined their growth properties and gene expression in defined, serum-free culture. The transgenic hepatocytes continued to overexpress human TGF-a mRNA and pepfide, and were able to proliferate without exogenous growth factors in primary culture, in contrast to nontransgenic mouse hepatocytes. In short-term culture the transgenic hepatocytes underwent 1 wave of DNA replication at 72â€"96 h in culture before senescing, similar to nontransgenic hepatocytes supple mented with epidermal growth factor. Constitufive expression of TGF-a rendered the transgenic hepatocytes unresponsive to further growth sUm ulation by exogenous TGF-a, as well as other mitogens such as epidermal growth factor and hepntocyte growth factor. However, it did not alter their sensitivity to growth inhibition by TGFfi1, 2 and 3. The addition of nicotinamide to the culture medium enabled both transgenic and epider mal growth factor-supplemented normal hepatocytes to replicate repeat edly and survive for 2 months in primary culture while maintaining differentiated traits. From these long-term primary cnitures of transgenic and nontransgenic hepatocytes, we established immortalized cell lines (designated TAMH and NMH lines, respectively) Both lines continued to express differentiated adult hepatocytic markers such as albumin, a-iantitrypsin, transferrin, and connexin 26 and 32 mRNAs, but also ex p1-eased mRNAs for the oncofetal markers a-fetoprotein and insulin-like growth factor II. Unlike the near-diploid NMH hepatocyte line, the trans genic TAMH hepatocyte line was quasl-tetrnploid, strongly expressed human TGF-a mRNA, and was highly tumorigenic in nude mice. Well differentiated hepatocellular carcinomas developed in nude mice given injecions of the TAMH line, and these appeared similar to the primary liver tumors seen in TGF-a transgemc mice with regard to histology and strong expression of mouse and human TGF-a, insulin-like growth factor H, and a-fetoprotein mRNAs. Our data show that TGF-a overexpression causes autonomous hepatocyte proliferation and contributes to neoplasia but that additional cellular alterations must occur for carcinogenesis. Inappropriate expression of insulin-like growth factor H may constitute one of these steps. The TGF-a transgenlc mouse hepatocyte line TAMH appears to undergo transformation in n similar manner to that of hepa tocytes overexpressing TGF-a in vivo, and should serve as an ideal system in which to study hepatocarcinogenesis.
Molecular Mechanisms of Hepatocarcinogenesis in Transgenic Mouse Models of Liver Cancer
Toxicologic Pathology, 2005
Overexpression of c-myc and transforming growth factor-alpha (TGF-α) has been frequently observed in human hepatocellular carcinoma (HCC), suggesting a pivotal role played by these protooncogenes in liver oncogenesis. In order to investigate the molecular events underlying human hepatic malignant transformation, we have generated c-myc and c-myc/ TGF-α transgenic mice that are prone to liver cancer. These transgenic mice develop HCCs with different incidence, kinetics and histopathological features. Indeed, co-expression of c-myc and TGF-α transgenes results in a dramatic synergistic effect on liver tumor development when compared with respective single transgenic lines, including a shorter latency period and a more aggressive phenotype. The more malignant histopathological features characteristic of c-myc/ TGF-α HCCs are the result of the increased proliferation and reduced apoptosis in this model of liver cancer when compared with single parental lines. Accordingly, c-myc and c-myc/ TGF-α transgenic mice display a different molecular pathogenesis of HCC. Importantly, the genetic and molecular mechanisms that are involved in c-myc and c-myc/ TGF-α liver cancer development are major oncogenic events in human hepatocarcinogenesis, indicating that these mouse models represent a useful tool to dissect and elucidate the molecular basis of human HCC.